Equilibrium Ligand Binding to the Human Erythrocyte Sugar ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc. Vol. 262, No. 12, Issue of April 25, pp. 5464-5475,1987

Pnnted m U.S.A.

Equilibrium Ligand Binding to the Human Erythrocyte Sugar Transporter EVIDENCE FOR TWO SUGAR-BINDING SITES PER CARRIER*

(Received for publication, June 23, 1986)

Amy L. Helgerson and Anthony Carruthers$ From the Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Equilibrium [SH]cytochalasin B binding to class I sites of human red cell membranes (the sugar transporter) was examined in the presence and absence of intracellular or extracellular sugars known to interact with the transport system. D-Glucose, a transported sugar, is without effect on cytochalasin B binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. Eth- ylidene glucose and maltose (reactive but nontransported sugars) inhibit cytochalasin B (CCB) binding when present either outside or inside the red cell. Inhibition by intracellular sugar (Si) is of the simple, linear competitive type. Inhibition by extracellular sugars (So) is more complex; the Kdcap,,, for cytochalasin B binding increases in a saturable fashion with [So]. These observations are compared with the predictions of the one-site, alternating conformer model and the two-site model for substrate binding to the sugar transporter, X. The experimental results are inconsistent with the one-site model but are explained by a two-site model in which the ternary complexes of So*X*Si or So*X*CCBi exist and where the binding sites for So and Si display negative cooperativity when occupied by nontransported substrate and little or no cooperativity when occupied by the transported species, D-glUCOSe.

Human erythrocyte sugar transport is mediated by an integral membrane glycoprotein (the sugar-sensitive, cytochalasin B-binding protein) of approximately 55 kDa molecular mass (1, 2). Although the simple one-site, mobile, or alternating conformer carrier model for facilitated diffusion (the classic carrier model) has been demonstrated to be an inadequate description of red cell sugar transport (for review, see Ref. 3), the available evidence supports the view that only one substrate binding site exists in the transport mechanism at any point in time (3-5).

A number of studies employing nontransported inhibitors of erythrocyte hexose transfer with high specificity for either cis- or trans- (influx and efflux) substrate binding sites have strongly suggested that occupancy of a cis-substrate binding site displaces inhibitor from the trans-site (5-7). For example, studies with purified sugar transporter ( 6 ) and intact red cells

* This work was supported by National Institutes of Health Grant R01 AM36081-01 and NIH Biomedical Research Support Grant SO7 RR0571. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Dept. of Biochem- istry, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01605.

( 7 ) demonstrated that phloretin, a nontransported inhibitor that reacts with an extracellular site on the transporter (4), competitively displaces cytochalasin B from a site that is exposed at the interior of the cell. Similarly, studies with erythrocyte membranes stripped of peripheral membrane proteins show that ethylidene glucose (a nontransported sugar with high, but not exclusive, specificity for the external substrate-binding site (8)) competitively displaces cytochalasin B from the internal site (5). These findings are consistent with the one-site carrier model for sugar transport (3).

Recent studies of the intrinsic tryptophan fluorescence of both purified sugar transporter and of transporter in red cell membranes stripped of peripheral membrane proteins have challenged the view that only a single substrate binding site is found on the transporter at any point in time (9,lO). These studies provided strong evidence for the simultaneous existence of at least two sugar-binding sites. In order for these findings to be compatible with the observations of cis-inhibition of occupancy of the trans-site by cytochalasin B, the two sites of the two-site carrier must display negative cooperativity. Occupancy of the cis- (or trans-) site must reduce the affinity of the trans- (or cis-) site for substrate.

This current study develops simple predictions for the one- and two-site models for substrate binding to the sugar transport molecule and demonstrates, by measuring equilibrium [3H]cytochalasin B binding to transporter in its native envi- ronment (the red cell membrane), that the one-site model is an inappropriate model for this system. Moreover, our findings support the view that cis- and trans-substrate binding sites display substrate-dependent, negative cooperativity when occupied by nontransported substrate but little or no cooperativity when the substrate is the transported molecule, D-glUCOSe. These findings are important, for they may explain why simple, one-site models for erythrocyte sugar transport have been unsuccessful in predicting the complex catalytic features of red cell hexose transfer (3).

EXPERIMENTAL PROCEDURES

Materials-[’H]Cytochalasin B and 3-O-[“C]methylglucose were purchased from New England Nuclear. 4-6-0-Ethylidene-a-glucopy- ranose (ethylidene glucose) was purchased from Aldrich. Maltose, D- glucose, mannitol, cytochalasins B and D, and phloretin were purchased from Sigma. The remaining reagents were purchased from Fisher and Sigma. Ethylidene glucose (EG)’ was >97% pure according to Aldrich Quality Control Analysis. We verified this by paper chromatography according to the method of Baker et al. (8). A major contaminant of ethylidene glucose is D-glucose (5, 8). Chromatogra- phy of 0.5 g of this batch of ethylidene glucose indicated that D- glucose was nominally absent (CO.1 pg).

Solutions-Wash solution consisted of 150 mM NaCl, 5 mM

The abbreviations used are: EG, ethylidene glucose; RBC, red blood cell; CCB, cytochalasin B.

5464

The Glucose Transporter Contains Two Sugar-binding Sites 5465

TrisHC1,2 mM EDTA, pH-adjusted to 7.4 using 1 M Tris base. Lysis medium contained 10 mM Tris-HC1,4 mM EDTA, pH-adjusted to 8.0 using 1 M Tris base. Stock cytochalasin B and cytochalasin D soh- tions were made in dimethyl sulfoxide. Phloretin stock solutions were made in ethanol. [3H]Cytochalasin B was supplied in ethanol. The final ethanol and dimethyl sulfoxide concentrations of all solutions were never *.I%. Control experiments indicated that these concentrations of carrier were without significant effect on ligand (cytochalasin B, sugars, etc.) binding to the carrier.

Red CeuS and Pink Red CeU Ghosts-Red cells were collected from whole, freshly outdated human blood as described previously (10). Pink erythrocyte ghosts were prepared from washed red cells by hypotonic lysis of red cells in lysis medium followed by four successive centrifugation/wash cycles (using lysis medium) and were resealed in wash medium (10).

[3H]Cytochalnsin B Measurements with Ghosts and RBCs-Equi- librium [3H]cytochalasin B binding to ghosts and RBCs was measured as described previously (12). This method is essentially that of Gorga and Lienhard (5). Briefly, 50 pl of RBCs or ghosts on ice (hematocrit = 5-25%) which had not been previously exposed to sugars or cytochalasin B (unless otherwise stated) were mixed with 50-200 pl of ice-cold [3H]cytochalasin B-containing solution (5-5000 nM cytochalasin B f sugars) in 1.5 ml-microcentrifuge tubes and incubated for 0-60 min. The tubes were then centrifuged at 4 "C for 1-10 min in an Eppendorff Bench Centrifuge and 2 X 20 ~l of the supernatant sampled and counted for activity. Parallel "blank" measurements were made with 50 pl of wash solution in place of ghosts or RBCs, and the difference between supernatant activities in blank and membrane runs was taken as binding. Free and bound cytochalasin B concentrations were then calculated. Light-scattering measurements made using the Coulter Electronics Submicron Particle Analyzer indicated that more than 99% of the membranes were pelleted under these conditions. Supernatant examined by phase-contrast micros- copy contained no visible (M.1 pm) particles. Binding was independent of centrifugation time provided that centrifugation was for 30 s or greater. Shorter centrifugation times (<30 s) resulted in the reten- tion of particles in the supernatant and a corresponding reduction in the amount of apparent binding detected. Control measurements made to determine the cytochalasin B binding properties of the Eppendorff tubes indicate that 101 * 1% of the [3H]cytochalasin B added to the tubes is recoverable from the bulk suspension present in the tubes both in the absence and presence of membranes (50 pl). Binding to the tubes is not promoted by the presence of sugars or cytochalasin D. Indeed, identical binding results are obtained by sampling the cell suspension prior to centrifugation (as a blank). Corrections for quenching of scintillation cocktail efficiency by cellular hemoglobin are eliminated by using saline (cell-free)-containing tubes as blanks. To be certain that the binding assay was reliable, we measured cytochalasin B binding to red cell ghosts using three different procedures: 1) the above procedure, 2) equilibrium dialysis using a microequilibrium dialysis apparatus (Hoeffer), and 3) counting the membrane pellet obtained upon centrifugation of the membrane suspension in [3H]cytochalasin B solution. With the latter procedure, corrections must be made for medium trapped in the pellet (cytochalasin B rapidly equilibrates with the interior of cells). This was achieved by making parallel runs in which 50 mM 3-O-["C]methylglucose was substituted for cytochalasin B. 3-0-Methylglucose is not metabolized by red cells and, due to its transport by the sugar transfer mechanism, may be used to estimate both intra- and extracellular water spaces. In this instance, where unsealed ghosts were used, equilibration of extra- and intracellular water by 3-0-methylglucose is extremely rapid ( 4 0 s, not shown). Cytochalasin B binding was measured in the presence and absence of maltose. Table I summarizes our findings. Binding is independent (+3% sampling error) of the method of binding determination.

All cytochalasin B solutions were made in wash medium. In experiments where sugars were added to displace cytochalasin B from ghosts or RBCs, control mns contained an equivalent concentration of mannitol, a sugar that neither reacts with the transport system nor displaces cytochalasin B from sugar-sensitive cytochalasin B binding sites (7). In some experiments, intact RBCs were preloaded with 100-500 mM D-glUCOSe by incubation in wash solution containing 100-500 mM D-glucose for 3 h at 37 'C. Turbidimetry measurements indicate that red cells are equilibrated with extracellular D-ghCOSe following this period of incubation. As a control, RBCs were also incubated with 100-500 mM mannitol for 3 h at 37 'C. Following loading, the red cells were washed twice in 200 volumes of ice-cold wash solution (containing 100-500 mM mannitol) to dilute the estra-

TABLE I Cytochalasin B binding to red cell ghosts

Method

~~ ~ ~

free/bound'

0 0.17 f 0.02 0.18 f 0.01 0.18 f 0.02 1 0.23 f 0.02 0.24 f 0.02 0.23 k 0.02

16 0.46 +- 0.03 0.45 f 0.06 0.45 f 0.03 166 0.62 f 0.05 0.60 f 0.07 0.61 * 0.04

Scatchard analysid

Equilibrium dialvsis Pelleted counts Loss of supernatant counts

Final concentration of maltose (mM) added to the binding assay where the concentration of class 1 sites (transporter) was 1.44 p M (5.6 X 10' cells in 100 pl) and the total ICCB] 100 nM. Cytochalasin D was present at 10 FM.

Cytochalasin B binding determinations by equilibrium dialysis for 48 h.

Cytochalasin B binding determinations by counting the activity obtained in the pelleted membranes.

Cytochalasin B binding determinations by counting the disappearance of activity from the supernatant upon centrifugation (the urocedure emuloved throughout the rest of this study: see "Emeri- mental Proceiurisn).

- _ .

e The measured free lCCBlhund ICCB1. Each result is the mean of two or more duplicate estimates.

(5.6 X 10' cells in 100 p l ) .

presence of 10 PM cytochalasin D.

ence of 10 p~ cytochalasin D. Temperature, 4 "C.

L~

'Scatchard analysis of cytochalasin B binding to red cell ghosts

8Calculated K1,.,,,, (in nM) for cytochalasin B binding in the

Calculated B , (concentration of binding sites, /IM) in the pres-

cellular D-glucose concentration to 125 p~ and then used immediately for cytochalasin B binding measurements. If binding to these cells was measured in medium containing <500 mM D-glUCOSe, the osmotic difference between cytosol and medium was adjusted to zero by including an appropriate concentration of mannitol in the external medium. This avoided hypotonic lysis of sugar-loaded red cells. Pre- loading ghosts with sugars was not necessary because the membranes were rendered leaky by omitting the resealing step (10) and by exposing the ghosts to a series of freeze-thaw cycles. Control experiments show that inhibition of cytochalasin B binding in ghosts at 1 min by addition of 100 mM D-glucose is identical to that measured at 30 min. Moreover, the ghosts are osmotically inactive (the reflection coefficient for sugars used in this study was too small to measure). Binding assays were performed at 24 "C or on ice (4 "C). Measure- ments of the time course of cytochalasin 3 binding to red cells and ghosts indicated that binding reaches equilibrium within 1 min of mixing membranes with cytochalasin B (see Fig. 3).

Turbidimetry Measurements-Sugar uptake by and the osmotic behavior of red cells and red cell ghosts were monitored by turbidimetry as described previously (10-12).

Analytical-Cell counts were made using a Coulter Electronics Coulter Counter, and light-scattering measurements were made using a Coulter Electronics Sub Micron Particle Analyzer.

Determination of Binding Constants-Under conditions where multiple components of binding could not be resolved by experimental manipulation, the method of successive approximation was employed (10). This is best illustrated by cytochalasin B binding to intact cells in the presence of 10 p~ cytochalasin D (see Fig. 5A). Binding to Hh is assumed to be a linear function in [CCB]. This assumption is supported by plots of bound versus free [CCB J in which cytochalasin B binding to red cells is largely a linear function of [CCB] superimposed upon a small saturable component of binding. The coefficient bound/free for binding of cytochalasin B to Hb is a constant (km) that is independent of [CCB] and, when plotted in Scatchard form, runs parallel to the 3c axis and intersects the y axis at km. km was estimated using an iterative procedure by first assuming that km is that value of bound/free [CCB] measured at the greatest determined

5466 The Glucose Transporter Contains Two Sugar-binding Sites

value of bound [CCB] (this value is an overestimate). Kl(,,, for saturable cytochalasin B binding was then estimated and used to refine the value of k m . The process is repeated until the estimated parameters (Kl(*) and k m ) change by < I part in lo6 and/or the experimental Scatchard plot is optimally fitted by the theoretical curve. The validity of this approach is illustrated by the fact that cytochalasin B binding in Hb-free ghosts is consistent with a single saturable component of binding (in the presence of cytochalasin D).

THEORY

The One-site System-Competitions between cytochalasin B and transported sugar for binding to the one-site (alternating conformer or mobile) carrier may be described by Scheme 1.1.

XCCB k-, k, JI k-3 41 C C B + X i + S i T X S i B

where X is unoccupied carrier, CCB is cytochalasin B, and Si and S, are intracellular and extracellular sugar, respectively. The empty carrier, X, undergoes isomerization to form X., (carrier with substrate binding site exposed to the cell's exterior) and Xi (carrier with an inward-facing substrate-binding site). As sugar transport is passive, Constraint:

klk--2k3k--6 = k-lkzk-ak5

k1Kok-6 = k-lKik5

where k-* /k2 = KO and k--3/k3 = Ki. This prevents transport against the gradient. Making the simplifying assumption that segments A and B of the scheme shown above are in rapid equilibrium (ie. transport and X isomerization are rate-lim- iting), the steady-state solution with respect to cytochalasin B binding is readily obtained by the method of Cha (13). Defining the following: A = [X.,] + [X. So], B = [Xi] + [X. Si] + [X.CCB], and [X,] = [total carrier] = A + B,

where K l = k - 4 / k 4 , we can derive the steady-state solutions from the following simplified King-Altman diagram.

f l k l

I f2k-1 11 I' A B

f 4k5

f 5k-5

The concentrations of the various complexes in the steady state are given by

where D = k - , f 2 + k-,f5 + k , f l + k5f4, [X.CCB] f 3 B - f 3 l k l f l + k5f4) - = ~

[Xtl [Xtl - D

___- [CCBI - [CCB] D

or, in the form suggested by Gorga and Lienhard (5),

[X.CCBl f 3 ( k l f l + k s f 4 ) [ X t l

where [CCB]/[X.CCB] is the ratio of free to bound [CCB] ( W ) .

When k-, = k5 = 0 (i.e. the sugar is nontransportable),

where D = k l fl + k- , f2 . This simplifies considerably to

" [CCB] [CCBID

[X.CCB] - f 3 k l f l [ X t ]

With nontransported sugar outside, cytochalasin B binding is competitively inhibited by So. For example, if k 1 = k- l , then in the absence of sugar, [x] = [Xi]. If nontransported sugar is then progressively added to the exterior of the cell, [X,] will be depleted due to the loss of [X,] to X. So. In the presence of cytochalasin B at a concentration that equals K1, but in the absence of sugar, one-half of the internal sites will be occupied by cytochalasin B, but by depleting [Xi] by forming X.CCB, a redistribution of carrier occurs such that [X.,] = [X,] = [X-CCB] = [X,]/3. Under similar circumstances but with the addition of nontransportable So([S , ] = KO) , [Xi,] (total concentration of carrier with intracellular facing sites) falls to 50% of [X,] due to the formation of X-So, thus [X.CCB] is reduced to [Xt]/4.

With transportable sugars, the situation becomes more complex, cytochalasin B binding becoming dependent upon the relative values of k l , k + , k5, and k-5. For example, if we assume that sugar-loaded carrier undergoes isomerization five times more rapidly than unloaded carrier and that transport is symmetric (i.e. KO = Ki, k, = k"5 = &fold k l , k l , remem- bering that klK0k-, = k-1Kik5) , then the presence of sugar at one side of the membrane will have a profound effect on the distribution of carrier forms. In the absence of sugar and cytochalasin B, [X,,] = [X,]. In the absence of cytochalasin B but under conditions where [So] = [Si], the carrier is again equally distributed between each face of the membrane. How- ever, in the presence of So (and absence of Si and cytochalasin B), a redistribution of carrier occurs, resulting in an increase in [X,] and decrease in [X.,]. Thus, if cytochalasin B is present, the net effect of raising [ S o ] is to increase the available inwardly oriented carrier and, hence, increase [X. CCB]. Qualitatively similar results are also obtained if the transfer mechanism is asymmetric (e.g. KO < Ki). However, absolute values of w are strongly dependent upon the values of rate constants employed. In view of this, we have adopted those values calculated by Lowe and Walmsley (141, who recently demonstrated that at 0 "C, the asymmetry in binding constants (assuming a one-site model, K,/Ki) is 0.75. The results of our calculations are shown in Fig. 1.

The Two-site Model-Competitions between cytochalasin B and nontransported (but reactive) sugar for binding to the two-site carrier may be described by Scheme 2.1.

XSi Si + X + CCB XCCB + + + S.. S,

The Glucose Transporter Contains Two Sugar

where a is a constant related to the cooperativity of the substrate binding sites (if a < 1, the sites show positive cooperativity; if a > 1, the sites show negative cooperativity), and the remaining symbols have the same meaning as per the one-site model. The major distinction of this scheme is that the carrier can exist as a ternary complex of X. So. Si or

At any substrate concentration (and assuming rapid equilibrium kinetics), the concentration of bound CCB ([X. CCB] + [S,.X.CCB]) is given by

X*So.CCB.

3

w

( L

1-

where y is (1 + [S,]/Ko + [Si]/Ki + [So][Si]/aKoKi]/{l + [S,]/aK,). Or, in the form suggested by Gorga and Lienhard (51,

The ratio of free [CCB] to bound [CCB] ( w ) is, therefore, linearly dependent upon y at any fixed [CCB]. However, y is also dependent upon the cooperativity constant a. If a = 1 and [Si] = 0, then y = 1, and extracellular sugar is without effect on CCB binding to the carrier (Fig. 2). If a > 1 and [Si] = 0, then y > 1 (y = {KO + [So]]/(Ko + [S ,] /a) ) , and w will increase with [So] in a fashion dependent upon the degree of saturation of the external sites by So (Fig. 2). In the absence of So, y = {I + [Si] /KiJ , and both y and w increase linearly with [Si] (Fig. 2). In the presence of both Si and So, when a = 1, y and, therefore, w increase linearly with [Si] (Fig. 2). If a > 1, the relationship between y and [SI is more complex and consists of both linear and saturable components (Fig. 2).

With transported sugars, e.g. D-glucose, the binding scheme (Scheme 2.2) is more complex.

CCB.X + So CCB.X.S,

lK1 4 ]‘ CCB CCB

+ + x + s, .K, xs,

+ 1 Ki

s;x + so ’ llK0 7 Si.XS”

binding Sites

ONE SITE

/ a / /

100 [SI mM

5467

FIG. 1. Predictions of the one-site model for cytochalasin B binding to the sugar transporter. Ordinate, freebound [cytochalasin B] (w). Abscissa, sugar concentration (mM). Conditions are [X,] = 1 PM, K , = 140 nM, [CCB] = 50 nM. The transport mechanism is presumed to display asymmetry in transport but little intrinsic asymmetry for substrate ( K O = 10 mM, Ki = 13.33 mM, k, = 1113,k-, = 90, kl = 12.1, k-, = 0.73 s-’). These values are obtained from a recent one-site analysis of the red cell transport mechanism at 0 “C (14). Results for transported sugars are given by curues a, c, and e. Curve a illustrates the effects of Si (0 So) on w. Curve c illustrates the effects of So (0 Si) on w. Curve e shows the effects of Si and So ([Si] = [S,]) on w. The effects of nontransportable S on w are shown by curves b, d, and f. Curve b shows the effects of Si alone on w, curve d the effects of So on w, and curue f represents conditions in which both So and Si are present ([So] = [Si]).

TWO SITE (non-transported sugar)

(transported sugar)

Here, the reactions Si.X w S.Xt, X.S, H Xt.S and Si.X.S, S . Xt. S represent the formation of intermediates associ-

ated with the process of translocation. For example, if we adopt the two-site, substrate-gated, water-filled pore model of Naftalin and Holman (15) (the existence of a water-filled pore is supported by recent hydrogen exchange studies (IS)), the carrier form Si -X represents carrier with substrate bound at the internal site and the form S .Xt represents carrier with substrate within the carrier pore. The intermediate S.Xt can still bind substrate at the external (but not at the internal) site to form S f Xt. S or, at subsaturating [So], can undergo

0 100 [SI mM

FIG. 2. Predictions of the two-site model for cytochalasin B binding to the sugar transporter. Ordinate, freebound [cytochalasin B] (w). Abscissa, sugar concentration, mM. Conditions are [X,] = 1 phf, Kl = 140 nM, [CCB] = 50 nM. The transport mechanism is presumed to display asymmetry for transported species ( K O = 1 mM, Ki = 5 mM; see Ref. 14). Accordingly, using the solution for Scheme 2.2 (see “Theory”), k-,/k, = 1 mM, k3/k-3 = 5 mM, k-,/k, = 0.14 +M, kJk-1 = 5, V I = uq = U - 1 = u-p = u3/2 = ~ - ~ / 2 = 1.33 X lo3/ s. Results are shown for transported (lower graph) and nontransported sugars (uppergraph, u1 = u2 = u g = 0). The substrate conditions are both Si and So present ([Si] = [Si] (a-c) ) , So present (0 Si ( d - f ) ) , and Si present (0 So (g-i)). Results are also shown for LY values of 4 (negative cooperativity; a d, and g), 1 (no cooperativity; b, e, and h ) and 0.25 (positive cooperativity, c, f , and i).


isomerization to form Xt. S where the internal binding site is now accessible to intracellular substrate but the external site is lost. Here, the substrate S may be envisaged as having progressed so far along the pore as to resemble the intermediate form produced by X. So. Thus, an efflux cycle (in the absence of So) is given by Si + X (both sites accessible) + Si. X (external site accessible) + S.Xt (external site accessible) + Xt. S (internal site accessible) + X. So (internal site accessible) + So + X (both sites accessible). Exchange transport is envisaged to involve the simultaneous movement of two sugars through the pore. In this state, the pore being doubly gated by substrate is opened maximally and permits the passage of two sugar molecules. As pointed out by Naftalin and Holman (15), this is consistent with the observed lower activation energy for exchange than for net transport. Of course, this is only one of a number of possible models. In order to simplify the solution for transport via the two-site model, we attempted to reduce the translocation steps to a minimum.

We have assumed (for simplicity) that nontransported sugar cannot penetrate the pore for steric reasons. In this instance, the solution (with respect to cytochalasin B binding) is given by Scheme 2.1. Furthermore, in order to simplify the algebra, we have assumed that substrate binding is rapid with respect to translocation steps. The validity of this assumption is not yet tested. The solution to Scheme 2.2 is presented below and was derived by a combination of the method of Cha (13) and by addition of multiple lines connecting two corners (17). This solution remains to be further reduced into parameters that are more readily experimentally determina- ble.

W = [CCBI/[CCBbtI = [CCB]('$A + ' $ A ' ) / ( f i @ A + f - r ' $ A ' ) [ x t ]

where '$A = u-If-1 + v-Zf-2 + U - 3 f - 3

'$A' = ulfl + u2f2 + u3f3

where vl-v-3 are translocation rate constants shown in Scheme 2.2.

f l = [ s i l / K ; / D ~ ; f z = [ & ] / K , / D A

f 3 = [ s . ] [ S i ] / a K , K i / D A , and

f 4 = I[CCB]/Kl + [ S ~ ] [ C C B I / I X K ~ K I ) / D A

where

DA = 1 + [S , ] /Ko + [ S J / K i + [CCB]/Kl

+ [So][Si]/aKoK; + [ S ~ ] [ C C B ] / C Y K ~ X ~

where the various dissociation constants (KO, etc.) are given in Scheme 2.2).

f - 1 = {k-lk-4(k-z + k3) + k-zk-3k--l[Si]}/D~,

f-z = {k-lk-4(k-z + k3) + kzk3k-4[So])/D~'

f - 3 = {klk-3k-4[Si] + k-lk-4kz[SoI + ~ - ~ ~ - ~ ~ Z [ S , I [ S ~ I } / D A '

f - 4 = {[CCBJk,(klk3 + klk-z + k z k 3 [ S o ] ) ) / D ~ .

where DA. = {k-lk-d(k-z + k3) + k-Zk-&4[Si]]

+ {k-lk-,(k-z + k3) + kzk3k--l[SolI

+ {klk-ak-4[Si] + k--lk-~kz[Sol + k--~k--lkz[Sol[Sil}

+ I[CCBIk4(klk3 + klk-z + kzkJSol)l

and where the various rate constants k1-k-, are shown in

Scheme 2.2. [X,] = total transporter concentration and [CCBb,] = total concentration of bound CCB.

The predictions of this complex model require assumptions as to the relative magnitudes of various rate constants in- volved in translocation. As these are not known, we have adopted values that do not contradict the passive nature of transport and that reflect the inherent asymmetry of transport. These requirements are

Ki/Ko = UlklU-z/U-lk-1Uz

aKo/@K0 = u - ~ v J u ~ u - ~

~uKi/@Ki = u - ~ u ~ / u Z U - ~

K.$KJK$K, = V-1uz/U1U-2

where PKo = k- , /kz; pKi = k3/k-, . If a = p, this means that V - l U J = V1U-3; V - z U s = U2U-3; U - I U ~ = U 1 U - z

The major determinant of catalytic asymmetry in transport is, therefore, the isomerization process (S.Xt w X+.S); i.e. K,/Ko = kl/k-l. Fig. 2 illustrates the predictions of the model. The parameters KO and Ki were arbitrarily assigned those values recently determined by rapid quenching techniques at 20 "C (approximately 1 and 5 mM, respectively (14); kl/k-l is therefore 5, and v 3 and u - ~ were arbitrarily assigned values 2- fold greater than vl-v-z in order to reflect 2-fold greater fluxes in the equilibrium exchange condition than during net exit (14). Similarly, ,!?KO = 1, PKi = 5, and k- , /k , = 0.14 X lo+. The absolute magnitude of the individual rate constants is unimportant provided that the relative magnitudes are main- tained. In the absence of transport ( v l = u p = us = 0), the above solution provides values identical to those produced by the simple two-site scheme (2.1). However, subtle quantitative differences are found when transport is permitted to proceed (see Fig. 2). The most important finding is that if negative cooperativity exists between the external sugar-binding site and the internal cytochalasin B binding site, a plot of w versus [So] is saturable whether or not transport occurs.

Conclusions-Figs. 1 and 2 summarize the predictions of the one- and two-site models for sugar-sensitive cytochalasin B binding to the erythrocyte hexose transfer system. Using recent estimates of the various rate constants for the one-site system (14), the one-site model predicts the following with respect to plots of w versus [So]. 1) If So is nontransportable, w is directly proportional to [So], but over the range of [SI of 0-80 mM and KO of 10 mM the effect on w is an increase of only 1.4-fold. 2) If So is transportable, w is slightly reduced by [So], but the effect would be extremely difficult to measure.

In the presence of So and Si ([So] = [Si]), whether S is transportable or nontransportable, w increases linearly with [SI at high [SI. However, for transportable S, the increase in w produced by [SI is less than that produced by [Si] alone.

The two-site model predicts the following. 1) If a = 1 (no cooperativity), then So is without significant effect on w. 2) If a > 1 (negative cooperativity), w increases in a saturable fashion with [So] with half-maximal effects observed when [So] = aK,. 3) If a < 1 (positive cooperativity), w falls (by approximately 50%) in a saturable fashion with increasing [So] with half-maximal effects observed when [So] = aK,.

In the presence of So and Si { [So] = [Si]) 1) if a = 1 (no cooperativity), then w is directly dependent upon [SI; 2) if a > 1 (negative cooperativity), w increases in a saturable fashion with [So] and linearly with [Si]; and 3) if a < 1 (positive cooperativity), at low [SI w first falls with increasing [SI and then increases in a linear fashion with further increases in [SI. This latter effect is probably difficult to detect experimentally.


These predictions are strict and facilitate the distinction between the one- and two-site carrier models for sugar transport.

Determination of a, KO, and Ki for the Two-site Model- The following procedures permit the calculation of a and KO for the two-site model with no transport from intact red cell cytochalasin B binding data. In the absence of Si, binding to the transporter is described by

”-

7 s - 7 . a - 1 a - 1

where

7 s = - - - - w’ - fc Kl7[XtI 0 - f, Kl[XtI

- - 7

where w’ = free/bound [CCB] in the presence of So, w = free/ bound [CCB] in the absence of So, and f, = [CCB]/[X,]. When [So] = -aKo, [S,]/(rs - yo) = 0. A plot of [S,]/(rs - 1) uersus [So] results in a single straight line with positive slope of 1/ (a - 1) and x intercept of -aK,. a can be calculated from the slope and KO from aK,.

Ki may be solved from binding data obtained with red cell ghosts in the presence of both Si and So. Fig. 2 illustrates that w increases linearly with [Si] and in a saturable fashion with [So]. The combination of these effects is a saturable curve superimposed upon a linear component. The simplest procedure for determining Ki is to “drop” the linear component of w uersus [SI at high [SI such that the y intercept is now w in the absence of S and the linear component is parallel to the original plot (Fig. 7). The x intercept of such a plot in the presence of So is -K(1 + [CCB]/K1).

Error Analysis-Fig. 3 illustrates that a only a single, major component of cytochalasin B binding to red cell membranes is detected in the presence of 10 p~ cytochalasin D. This component is the sugar-sensitive component previously class- ified as site I (see below and Ref. 7). Unless stated otherwise, all experiments were performed in the presence of cytochalasin D.

As the distinction between one- and two-site models for

BOUND CCB ilmoiilo‘* c*U*

FIG. 3. Components of [aH]cytochalasin B binding to human erythrocyte pink ghosts. Ordinate, [bound cytochalasin B]/[free cytochalasin B] in m ~ l / l O ’ ~ cells/mol/liter of incubation medium. Abscissa, bound [cytochalasin B] in pmol/1013 cells. Binding was measured over a range of cytochalasin B concentrations (5-2500 nM) in the absence (0, A ) and presence (0, B ) of 10 p~ cytochalasin D. In the presence of cytochalasin D, the data fall on a single, straight line (least squares analysis results in a correlation coefficient, R > 0.99) with [X,] of 2.59 pm~l/ lO’~ cells and K , of 150 k 9 nM. In the absence of cytochalasin D, the binding curve consists of two components: the component observed in the presence of cytochalasin D (curue B ) and a smaller component (C) with [X,] of 0.41 pm~l/ lO’~ cells and K1 of 36 nM. Component C was obtained by subtraction of curue B from A. Number of assays/point, 2. Temperature, 24 “C.

cytochalasin B binding rests upon the demonstration of nonlinear plots of 0 uersus [SI, it is necessary to consider factors (other than proposed mechanisms) that could produce such results. A major factor could be the method of determining [3H]cytochalasin B binding to membranes. The method we have adopted is to measure the disappearance of cytochalasin B from the supernatant of pelleted suspensions of cells and medium using cell-free blanks as controls. This means that upon addition of ligand-displacing sugar, bound [ligand] falls and free [ligand] increases. Thus, when calculating a theoretical w value, the calculations should take into account the increase in [CCB] that occurs upon addition of inhibitor. Figs. 1 and 2 do not correct for the effect of raised [CCB]. This could introduce errors into the interpretation of findings. Consideration of the solutions to Schemes 1.1 and 2.1 indicates that these errors are, in fact, small (<2%). Provided that the term [CCB]/[X,] is small (i.e. [CCB] << [X,]), the errors introduced by failing to recognize the increase in [CCB] upon addition of cytochalasin B-displacing ligand are negligible. For example, using ghosts at 20 “C under conditions where [X,] = 1.13 pM, [CCB],,I = 50 nM, K , = 140 nM, and [ethylidene glucose] = 0, we measure an equilibrium [CCB] free of 5.74 k 0.36 nM ( n = 6). w is therefore 0.129 f 0.01. When [EG] is increased to 100 mM, [CCB] rises to 21.5 f 0.9 nM (n = 6) and w is increased to 0.754 f 0.057. Thus, for the one-site model (and using the parameters calculated in Table 111), theoretical w at 100 mM EG and 21.5 nM CCB is 0.95, and at 100 mM EG and 5.74 nM CCB is 0.94. The error in failing to consider the increase in [CCB] from 5.74 to 21.5 nM is, therefore, -1%. Similarly, with the two-site model (and using the parameters calculated in Table 111), theoretical w at 100 mM EG and 21.5 nM CCB is 0.77 and at 100 mM EG and 5.74 nM CCB is 0.76. The error is, therefore, -1.8%. These errors are smaller than the experimental sampling errors.

A second potential source of error in studies with intact cells is binding of cytochalasin B to hemoglobin. Displacement of ligand from transporter by S would increase [CCBIfm and, therefore, the concentration of cytochalasin B bound to Hb, thereby reducing true w. Analysis of cytochalasin B binding to intact cells indicates that Hb binds cytochalasin B nonspecifically with a constant of 3 f 0.1 X 10‘ sites/cell/pM cytochalasin B (see Fig. 5A and also Ref. 7). This must be considered when calculating w for binding to site I in intact cells. Table I1 indicates that failure to correct for this effect significantly reduces w for binding to site I. Inadequate cor- rection for binding to Hb cannot, however, be the explanation for the saturable relationship between w and [So] in ghosts, for the ghosts are 98-99% free of Hb. Under these conditions (and at 5-15 nM CCBfme), [Hb.CCB] accounts for less than 1% of total cytochalasin B binding. Moreover, this could not explain the inability of D-gluCOSe to inhibit binding in intact cells where ethylidene glucose, maltose, and phloretin are potent inhibitors of binding (see below). Binding to residual Hb in ghosts cannot account for the linear relationship between w and [D-glucose] and the more complex relationship between w and [ethylidene glucose] or [maltose] (see below). In all experiments with intact cells (except Fig. 4), binding to Hb has been subtracted from total binding at the measured [CCBIfr, and, in principle, reflects binding to class I sites only.

The possibility also exists that the method of cytochalasin B binding determination is in error. We show in Table I that the binding results obtained by three different methods (including that employed in this study) are indistinguishable. Finally, with respect to nonlinear inhibition curves obtained using extracellular maltose, ethylidene glucose, and phloretin, it is possible that the maltose, ethylidene glucose, and phlor-


etin solutions employed in this study contain contaminants that interact with the transport mechanism. With ethylidene glucose, these contaminants cannot be sugars because within the limits of detection (<0.1 fig) the sugar was chromatograph- ically pure, as determined by paper chromatography. More- over, the inhibitions of Sen Widdas D-glucose exits in ghosts by ethylidene glucose and maltose (that concentration of sugar that reduces saturated exit by half) are indistinguishable (within experimental error) from those reported for inhibition of exit in intact cells using maltose and purified ethylidene glucose (see Table 111).

RESULTS

A major strategy we have employed is to measure [3H] cytochalasin B binding to red cells in the absence and presence of intracellular sugar f extracellular sugar. This was achieved in the following fashion.

TABLE I1 Errors introduced by failure to consider cytochalasin B binding to

hemoglobin Cytochalasin B binding to intact red cells at 4 “C was measured in

duplicate at 50 nM total cytochalasin B. [Hb‘CCB] was calculated from the measured free [CCB] as [Hb.CCB] = 2.26 [CCBIf where 2.26 is the measured concentration (pM) of Hb.CCB sites present in the aSSay/pM free [CCB]. [X. CCB] (the concentration of class I sites occupied by CCB) was calculated as [CCB],, - [Hb.CCB]. “True” w is the ratio of free CCB to CCB bound at class I sites. [Xt], the total concentration of class I sites, was 1.13 p~ and Kl(a,,,,l for CCB binding to these sites was 140 nM.

[EG] [CCB]? [CCB]b* [Hb.CCB] [X.CCB] “True” estimation Under-

* n f r . ,

mM nM 0 4.55 0.1 4.9 0.25 5.41 0.5 6.21 1 6.73 2.5 7.76 5 8.36 10 8.76

nM 45.45 45.1 44.59 43.79 43.27 42.24 41.64 41.24

nM 10.28 11.07 12.23 14.03 15.21 17.54 18.89 19.8

nM 35.17 34.026 32.36 29.76 28.06 24.7 22.75 21.44

0.1 0.109 0.121 0.142 0.156 0.184 0.201 0.212

0.129 0.144 0.167 0.209 0.24 0.314 0.368 0.409

% 29.0 32.1 38.0 47.2 53.8 70.7 83.1 92.9

Measured free [CCB]. Total bound [CCB].

With intact red cells, it is possible to mix sugar-free (extensively washed) erythrocytes with wash medium containing both cytochalasin B and sugar a t 4 “C and then to measure equilibrium [3H]cytochalasin B binding at 1 or 2 minutes by rapid centrifugation before any appreciable quantity of sugar penetrates the cells. Vmax for D-glucose uptake at this temperature (and in the absence of cytochalasin B) is 5.5 pmol/liter cell water/sec (14). The concentration of D-glucose within the cells at 1 or 2 min (in the absence of cytochalasin B) is therefore in the order of 0.3-0.6 mM. In the presence of cytochalasin B (a transport inhibitor), uptake is, in fact, lower than this. With nontransported but reactive sugars (e.g. maltose and ethylidene glucose), the concentration of sugar inside the cells at 1 and 2 min at 4 “C is negligible. Alternatively, red cells can be loaded with D-glucose (or ethylidene glucose, a more lipophilic species) by incubation at 37 “C for 2-4 h (8) and then exposed to sugar-free or sugar-containing medium at 4 “C. [3H]cytochalasin B binding may then be measured in the presence of intracellular sugar and presence and absence of extracellular sugar. These procedures permit the measure- ment of cytochalasin B binding to intact red cells under conditions where the external or internal sites are saturated with sugar.

Using extensively washed, unsealed ghosts that have been exposed to a number of freeze-thaw cycles to render their membranes highly permeable to sugars (7, 10, 12), [3H]~yto- chalasin B binding may be measured under conditions where both sides of the red cell membrane are simultaneously exposed to sugar.

Cytochuhin B Binding Sites-Cytochalasin B has been reported to bind to two (and possibly three) distinct classes of sites in red cell membranes and, in intact red cells, to bind nonspecifically to hemoglobin (7, 18). The membrane sites include the sugar transporter (site I, an integral membrane protein, KlcaPp, = M, sites/cell = 1.3-3 X lo5), cytoskeletal elements (site 11, peripheral membrane protein, KlcaPp, = M, sites/cell = 0.5-1 X lo5), and an as yet unidentified component (site 111, peripheral membrane protein, Kl(epp) =

M, sites/cell = 0-0.7 X lo6). Binding to site 11 (but not sites I and 111) is inhibited by cytochalasin D (19).

In this study, Scatchard analysis of equilibrium cytochalasin B binding to pink ghosts suggests the presence of only

TABLE 111 for sugar interaction with the human red cell sugar transport system

References to the appropriate studies are indicated in parentheses. D-glucose” Ethylidene glucose Maltose Phloretin

a& K; a KO aK. K; a KO aK. K, a K, aK, K; a KO

This study NDb

Fluorescence quenching Band 4.5 (20) Band 4.5 (9) 1.5 Membranes (10) 1.33

Band 4.5 (6) Membranes (7) Membranes (5)

Red Cells (3) Ghosts (10)

1.7 11

Cytochalasin B binding

Transport

15.9 1 ND 1.6 37.1 3.4 0.48 3.3 60 3.8 0.89 f 1.2 f 0.1 f 1.9 & 0.2 f 0.02 f 0.2 f 2.7 f 0.1 & 0.06

48 16 26.9 1.9 29.7 39.0 0.69 27.1

2.6 128 2.9 107

43 40-60

59

120

26

5-40 1.8’ 11w 11 2.3

f 0.2f

6-14d 4.7

2 0.6’

1.99 ND 37.1 0.054 f 0.1 * 3.4 & 0.002

0.96 1.00

24 2.4

0.24‘ 0.5

f 0.1‘

All parameters are in mM with the exception of phloretin data, which are in p ~ . The cooperativity factor a

ND, not detected in this study. e From Ref. 8.

From Refs. 8 and 31. e From Refs. 22 and 23. ‘A. Carruthers and D. L. Melchoir, unpublished results.

has no units.


two classes of binding sites (Fig. 3). Cytochalasin B binding is significantly reduced by the simultaneous presence of cytochalasin D (10 p ~ ) and is consistent with a single class of sites (site I) with Kl(app) of 150 nM and capacity of, 1.5 X 10' sites/cell (Fig. 3). The difference between control binding and binding in the presence of cytochalasin D is again consistent with a single class of sites (site 11) with Kl(app) of 36 nM and capacity of 2.5 X 10' sites/cell. Under the conditions employed in this study, we find no evidence for class I11 sites. This is illustrated by the observation that at 500 mM D-glucose, 10 p~ cytochalasin D, and a free [3H]cytochalasin B concentration of 5 nM, binding is inhibited relative to control (500 mM mannitol, 10 p~ cytochalasin D) by 96 f 3% ( n = 4). These findings are in good agreement with those of Lin and Snyder (18).

The remaining experiments described in this study were performed in the presence of 10 p~ cytochalasin D and thus relate to cytochalasin B binding to class I sites (the sugar transporter).

Time Course of Cytochalasin B Binding to Intact Red Cells and Red Cell Ghosts-Fig. 4 illustrates the time course of [3H] cytochalasin B ([CCB] = 100 nM) binding to intact red cells (f500 mM intracellular/extracellular D glucose) at 4 "C. Bind- ing equilibrium is achieved within 1-2 min of mixing both sugar-free and sugar-loaded red cells with medium containing cytochalasin B and remains unchanged for up to 30 min. Interestingly, extracellular D-glucose is without effect on cytochalasin B binding to red cells both in the presence and absence of intracellular D-glucose. On the other hand, intracellular D-glUCOSe is an effective inhibitor of [3H]cytochalasin B binding to the red cell, reducing the total concentration of occupied sites by 50%. In intact cells, nonspecific cytochalasin B binding to hemoglobin accounts for as much as 3-3.2 X lo5 sites/cell/pM cytochalasin B (7). At 100 nM cytochalasin B, as much as 33% of total binding is thus accounted for by nonspecific adsorption to hemoglobin, and assuming a Ki (app)

for D-glucose inhibition of cytochalasin B binding to class I sites of 15 mM (see below), binding to class I sites should be inhibited by 97%. If binding to intact cells in the presence of cytochalasin D is mostly accounted for by class I sites and hemoglobin, the expected inhibition of cytochalasin B binding to intact red cells at 500 mM intracellular D-glUCOSe is 45%, a value very close to the experimental findings. The one-site model predicts that extracellular D-glucose should reduce the

2 6 P ' l ! i i L 0 0 TIME MIN 30

FIG. 4. Time course of cytochalasin B binding to red cells and red cell ghosts and the effects of extracellular and intracellular sugars on binding. Ordinate, pmols of cytochalasin B bound to red cells (0, 0, 0, =) and ghosts (A, A). Abscissa, time in minutes. Binding to red cells was measured in the absence of D- glucose (o), the presence of 100 mM intracellular D-glucose (W), the presence of 100 mM extracellular D-glucose (O), and the presence of 100 mM D-glucose in both the intra- and extracellular media (0). Binding in ghosts was measured in the absence (A) and presence (A) of 100 mM D-glucose. Temperature, 4 "c. Centrifugation for 1 min. Cytochalasin D (10 pM) was present throughout. Each point represents the mean of duplicate assays. Free [[3H]cytochalasin B], 100 nM. Number of ghosts/ghost assay, 8.1 X lo'. Number of red cells/ red cell assay, 5.4 X 10'. Red cell data have not been corrected for cytochalasin B binding to hemoglobin.

inhibition of cytochalasin B binding by intracellular D-glucose at 4 "C. This is not observed experimentally.

Effects of Sugars on Cytochalasin B Binding to Red Cells and Ghosts-Fig. 5 illustrates the effects of intra and extracellular sugars on the concentration dependence of cytochalasin B binding (at 2 min) to red cells at 4 "C. These data are shown in Scatchard form and illustrate our method for analysis of cytochalasin B binding to hemoglobin (see "Experi- mental Procedures"). Again, D-glUCOSe is without effect on binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. On the other hand, ethylidene glucose is a potent inhibitor of binding when present in the external medium. The effects of extracellular ethylidene glucose and intracellular D-ghCOSe are restricted to increasing Kl(app) for cytochalasin B binding. Fig. 5 also shows that sugars (D-glucose and ethylidene glucose) act as competitive inhibitors of cytochalasin B binding to red cell ghosts. This is predicted both by the one- and two- site carrier models for transport. In order to test the models further, it is necessary to determine how sugars affect the

A

t m 8

0 0 3

20 0

:

bound CCB 3

FIG. 5. Effects of various sugars on cytochalasin B binding to red cells and red cell ghosts. A, Scatchard analysis of cytochalasin B binding to red cells at 4 "C. Ordinate and abscissa as in Fig. 3. Binding to intact red cells at 4 "C was measured in the absence of sugar (O), the presence of 500 RIM extracellular D-glucose (A), the presence of 500 mM extracellular ethylidene glucose (O), the presence of 500 mM intracellular D-glucose (e), the presence of 500 mM intra- and extracellular D-glucose (A), and the presence of 500 mM intracellular D-glucose and 500 mM extracellular ethylidene glucose (=). Binding assays were made over the cytochalasin B concentration range 5-2500 nM in the presence of 10 p~ cytochalasin D, and measurements were taken at 2 min. The data have been"corrected' for nonspecific binding to hemoglobin (5 pmol sites/l0l3 cells/pM cytochalasin B; see inset for Scatchard analysis of total (uncorrected) binding to red cells). Each point represents the mean of duplicate measurements. The inset shows the Scatchard analysis of uncorrected binding to intact red cells for binding in the absence of sugars. The curves are total binding (O), corrected binding by subtraction of binding to hemoglobin ( 0 , see "Methods"), and binding to hemoglobin (A). B, Scatchard analysis of cytochalasin B binding to red cell ghosts. Ordinate and abscissa as in Fig. 3. [3H]cytochalasin B binding was measured in the absence (0) and presence of 250 mM 1-0-methylglu- copyranoside (V), 250 mM ethylidene glucose (O), 250 mM maltose (O), and 250 mM D-glUCOSe (A). Each point represents the mean of duplicate estimates. In instances where only one point is shown (i.e. there is not an obvious pair of points) each point represents the mean of duplicates of two measurements. Temperature, 24 "C.


Kl(app) for cytochalasin B binding to red cells or to determine how the ratio of free uersus bound ligand (0) varies with sugar concentration. Fig. 6A illustrates the effects of increasing ethylidene glucose concentration on the concentration dependence of cytochalasin B binding to class I sites in red cell pink ghosts. The Scatchard plots demonstrate that ethylidene glucose increases the Kl(epp) for cytochalasin B binding to the membranes without significant effect on [X,]. However, a plot of Kl(app) uersus [ethylidene glucose] (Fig. 6B) illustrates that Kl(app) is not a simple monotonic function in [ethylidene glucose] but rather that K1(,,,,, increases in an abrupt, saturable fashion over the [ethylidene glucose] range 0-10 mM and linearly thereafter. This behavior is not predicted by the one-site carrier model but is accounted for by the two-site model if (Y > 1.

Similar curvilinear plots are obtained upon plotting w (free/ bound [CCB]) uersus [ethylidene glucose] (Fig. 7). Also in- cluded in this figure are results of binding studies using D- glucose and maltose. Maltose inhibition of cytochalasin B binding to pink ghosts is similar to that produced by ethylidene glucose. However, D-glucose, unlike maltose and ethylidene glucose (sugars that bind to the transporter but, for steric reasons, are not transported (8)), increases w monotonically with [D-glucose].

Fig. 7 also illustrates the effects of D-glucose, ethylidene glucose, maltose, and phloretin on w for cytochalasin B binding to intact red cells under conditions where intracellular sugar is nominally zero (4 “C). This is an important test for the following reasons. I) The one-site model predicts that for nontransported sugars w should increase monotonically with extracellular [sugar]. Using transported sugars, however, w should be unaffected by So. 2) The two-site model predicts that if the cooperativity factor, a, is greater than 1, w will increase in a saturable fashion with [ S o ] or, if a = 1, that w is unaffected by So. The findings demonstrate that D-glucose, is without effect on w but that in the presence of ethylidene glucose, maltose, or phloretin, w increases in a saturable fashion with [S,]. These results again indicate that the one- site carrier is an incomplete formalism for describing sugar- sensitive cytochalasin B binding to human red cell membranes. Moreover, they raise the intriguing possibility that with the two-site model, the two substrate binding sites are negatively cooperative when occupied by nontransported sug-

A B

2oK

,,- [Ed M 0

BOUND CCB 3

FIG. 6. Effect of ethylidene glucose on cytochalasin B binding to site I in red cell ghosts. A, Scatchard analysis of binding. Ordinate and abscissa as in Fig. 3. Binding was measured in the presence of increasing ethylidene glucose concentrations (from top to bottom, 0,2.5, 1,5, 25,50,100, and 250 mM ethylidene glucose). Each point represents the mean of at least two separate measurements made in duplicate. Temperature, 24 ‘C. The lines drawn through the points were calculated by least squares. B, effect of ethylidene glucose on Kl,app) for cytochalasin B binding to site I. Ordinate, Kl,aw), nM. Abscissa, ethylidene glucose concentration, M. Klc,, was calculated as -l/slope from least squares analysis of the data shown in A. The curue drawn through the points is of no theoretical significance.

ethylidene glucose

t 1.4 2 t 1.4

maltose

r 2.5 0-glucose

L r 1 2 phloretin

0 CII mM 2 5 0 0 n l pM 2 5 0

FIG. 7. Effects of various sugars and phloretin on the ratio of free/bound [cytochalasin B] (w) in red cells and red cell ghosts. Ordinate, w. Abscissa, inhibitor concentration in mM (or with phloretin, in p ~ ) . Data are shown for ethylidene glucose, maltose, D- glucose, and phloretin experiments; the inhibitor is indicated above eachgraph. Open circles represent data obtained from ghosts at 24 “C, and the filled circles represent data obtained from red cells a t 4 ‘C. Where the data points overlap, the data is represented by a half-filled circle. Number of duplicate measurements/point, 4 or more. In red cell experiments, the sugar was present (initially) only in the extracellular medium and with ghosts, both extracellular and cytoplasmic surfaces of the plasma membrane were exposed to sugars. With ethylidene glucose, maltose, and D-glucose experiments, the number of cells/assay was 3.9 X 10’. Free [cytochalasin B] was = 40 nM. With phloretin, the number of cells per assay was = 2 X 108 and the free [cytochalasin B] % 100 nM. Nonspecific binding to hemoglobin in intact cells has been subtracted from the red cell data. The curues are drawn by eye. With ethylidene glucose and maltose, an additional curue is drawn which represents the linear component of w uersus [SI. The significance of this curue is that the x intercept = -Ki(l + [CCB]/K,) for the two-site model (see “Theory”).

ars but fail to interact significantly when occupied by transported substrate.

Using these findings, we might predict that upon mixing sugar-free red cells with D-glucose, initially external sugar would be without effect on cytochalasin B binding to red cells but, as glucose penetrates the cells due to transport, cytochalasin B will be competitively displaced from the intracellular binding site. Conversely, occupation of the external site by maltose and ethylidene glucose will initially displace cytochalasin B from the internal binding site with little effect thereafter due to much lower uptake rates. Fig. 8 shows that if the experiments are performed at 20 “C where D-glucose transport rates are substantial, these predictions are confirmed experimentally.

DISCUSSION

The results of this study support the view that the sugar- sensitive cytochalasin B-binding protein (sugar transporter) of human erythrocyte membranes contains two substrate (sugar)-binding sites. For the transported sugar, D-glucose, these sites display little or no interaction. For nontransported sugars, these sites display marked negative cooperativity.

With the one-site model, maltose, ethylidene glucose, and phloretin would inhibit cytochalasin B binding to the internal isomer of the sugar binding site by “trapping” or stabilizing the carrier in the external isomer form, Xes.,, thus reducing the amount of internal isomer available for reaction with cytochalasin B. This inhibition of cytochalasin B binding should be of the linear, competitive type (see “Theory”). The results of this current study demonstrate that the inhibitions by these agents are of a more complex nature, closely resem-


I- t 0-

0 3 TIME HOURS

chalasin B binding to intact red cells. Ordinate, freebound FIG. 8. Time course of sugar-induced inhibition of cyto-

at 5 and 10 min (at 24 "C) in the absence of added sugar (V) and [cytochalasin B] (w). Abscissa, time in hours. Binding was measured

then, at 10 min (see arrow), D-glucose (0), maltose (A), or ethylidene glucose (0) was added from a stock of 1 M sugar to give an initial extracellular concentration of 100 mM. Data are also shown for the effects of addition of 100 mM D-glucose on binding at 4 "C (0). Each point represents the mean & S.D. of two measurements made in duplicate. Centrifugations were for 1 min. Data have been corrected for nonspecific binding to hemoglobin. Number of cells/assay, 3.9 X lo"; free [cytochalasin B], approximately 40 nM.

bling those predicted by a two-site model in which occupation of the external site reduces the affinity (increases K 1 ) of the simultaneously existent internal cytochalasin B-binding site for ligand by a factor a.

If the external sugar is a transported species (e.g. D-glucose), the one-site model predicts little effect of So on binding. No effect was observed in this study. The two-site model also predicts that external D-glucose is without effect on cytochalasin B binding provided that occupation of the external site by D-glUCOSe does not promote negative or positive cooperativity between internal and external sites. Thus, our experimental findings are in agreement with the predictions of the two-site model and only in partial agreement with the one- site scheme.

Comparisons with Previous Binding Studies-Assuming that the two-site model is an accurate description of cytochalasin B binding to the transporter, it is possible to obtain values of KO, K;, and a for the various sugars/inhibitors from the experiments made with intact red cells and ghosts (for details see "Experimental Procedures"). Table I11 summarizes these calculations for D-glucose, ethylidene glucose, maltose, and phloretin. The cooperativity factor increases in the order, glucose = 1 << ethylidene glucose 5 maltose << phloretin. In the absence of any effect of external D-glucose on cytochalasin B binding, KO for D-glUCOSe cannot be calculated. Table I11 also compares a&, and K , parameters from this study with those obtained in fluorescence quenching studies of purified transporter and red cell membranes and with those measured in studies of cytochalasin B binding to purified transporter and ghosts. In most instances the agreement is very good.

Most of the previous studies of cytochalasin B binding to red cell membranes and purified transporter have been performed at sugar levels of 5-500 mM (5-7, 18). It is unlikely that these studies could accurately detect the low aK, parameters (lower concentrations of sugar must be used), and the calculated inhibition constants for cytochalasin B binding would be weighted largely by ligand binding to the low affinity internal site. Assuming this was the case, the results, K j (app)

= 43-59 mM, 26 mM, and 120 mM for D-glUCOSe (5-7), ethylidene glucose (5), and maltose (6), respectively, are in good agreement with those detected in this study and fluorescence studies (9, 10). Indeed, the study with ethylidene glucose (5) suggested a value for KO of 26 mM. Using recent estimates of

the various isomerization and translocation rate constants for the one-site model (14) and this value for KO, [EG,] at 80 mM would produce a marginal (1.2-fold) increase in w (via the one-site carrier) and not the observed increase (5) of 6.2-fold. Each of these studies (5-7, 18) has reported a linear depend- ency of w or Kl(app) on [SI that directly extrapolates from high levels of [SI to W, when [SI = 0. We confirm this here for D- glucose but not for maltose, ethylidene glucose, or phloretin. The reasons for these differences are not related to the use of purified transporter (6) or alkali-washed ghosts (5) because substrate-induced tryptophan fluorescence quenching studies with these preparations indicate two substrate binding sites/ transporter (9 and 10, but see 20). The observations in this current study are independent of the method of binding assay (see Table I), seem not to result from detectable sugar impur- ities in the sugars employed, and do not result from nonequi- librium cytochalasin B binding or incomplete sedimentation of membranes.

Two previous studies of the effects of phloretin on cytochalasin B binding to purified transporter (6) and red cell membranes (7) reported inhibition of the linear competitive type, a result that was not confirmed in this study. The study with red cell membranes (7) is difficult to reconcile with our findings. However, a replot of Fig. 13 of Ref. 7 as w uersw [phloretin] indicates a clear deviation from linearity with saturation at higher phloretin levels, although the calculated a parameter from the data in Fig. 13 is substantially lower than our estimate of 37, and the KO parameter (1.8 pM) is 34- fold greater than the value we measure (54 nM). Studies with phloretin are complicated by the saturable binding of the transport inhibitor to membrane lipid (5 X lo7 sites/red cell (1:9 ph1oretin:lipid molar ratio), Kl(app) = 10-40 pM (21)). Thus, the equilibrium free phloretin concentration in these studies (including the current study) is reduced by binding to lipid to a greater extent at low total [phloretin] than at higher total [phloretin]. This results in nonlinear increases in free [phloretin] with total [phloretin] and therefore compromises data analysis.

The Ki(app) for phloretin inhibition of sugar entry (9 p~ (22)) is substantially greater than KicaPp) for inhibition of glucose exit and exchange (0.24-0.5 p~ (23)). These values are reliable because extremely low hematocrits were employed; thus, binding of phloretin by cells would have minimal effects on the bulk solution phloretin concentration. As the exit condition is more applicable to phloretin inhibition of cytochalasin B binding, the reported K,(app) for phloretin inhibitions of cytochalasin B binding of 23 (6), 5 (7), and 1.9 p~ (see Table 111) are substantially greater than that for inhibition of exit. Thus, problems resulting from phloretin binding to membrane lipid could result in overestimation of Ki(app) in cytochalasin B binding studies.

The findings of a recent proton NMR study of &D-glucose binding to erythrocyte ghosts have been interpreted to be consistent with the one-site model (24).

Comparisons with Transport Data-Table I11 summarizes the basic Michaelis Km(app) and inhibitory constants Kj(epp) for transport and inhibition of transport by the various sugars employed in this study. Considering glucose first, the parameter K; (from this study) is in excellent agreement with the values of Kmfepp) for glucose exit in cells and ghosts. Indeed, the value for Kj estimated in this study is much closer to Km(epp) for exit than previously estimated (5-7). With ethylidene glucose, we find very close agreement between aK, for ethylidene glucose inhibition of cytochalasin B binding and Ki(app) for extracellular ethylidene glucose inhibition of sugar exit. Previous estimates of aKo (5,9) which were assumed to be mediated by the high affinity external site are some 9-14-


fold greater than the observed Ki(app) for inhibition of exit by external ethylidene glucose. With maltose, the estimated aKo parameter is some 2-4-fold lower than for external maltose inhibition of sugar exit. However, previous studies (6) did not detect the low a K , parameter, but only the Ki parameter for maltose-inhibition of cytochalasin B binding. Both aK, and Ki for maltose are detected in this and fluorescence quenching studies (9, 10).

This current study and previous fluorescence quenching studies (9, 10) have generally supported the premise of asymmetry in sugar binding constants derived from transport studies. Moreover, they have demonstrated the existence of these two classes of sugar binding sites which, although predicted by transport studies, have not been detected in previous cytochalasin B binding studies.

Recent Transport Data-That transport condition analo- gous to measurements of sugar inhibition of cytochalasin B binding and measurements of sugar-induced fluorescence quenching with band 4.5 protein and red cell membranes is the equilibrium exchange condition ([Si] = [S,]). Recent estimates of the Michaelis constant for exchange transport are 12-17 mM (3). It has been reported, however, that two components of D-glucose exchange are detected with Km(app) of 2.3 and 26 mM (25). These values are very close to those detected in fluorescence quenching studies with band 4.5 protein and red cell membranes stripped of peripheral proteins (1.8 and 30 mM; see Refs. 9 and 10) and have been cited as evidence for negative cooperativity in sugar transport (25). This result (25), which required highly precise transport measurements, was not seen in a subsequent study (3).

Some, but not all, of the difficulty in interpreting red cell D-ghCOSe transport data results from the anomalously high Michaelis constant for sugar efflux into sugar-free medium (K'", zero-trans efflux). At 20 "C, the ratio of Michaelis and velocity constants for zero-trans efflux and influx ( P / W i and V/P) are approximately 15 and 5, respectively (3). For a passive transport system (such as red cell sugar transport), the Haldane requirements are that these ratios are identical. In a recent study of the initial rate of zero-trans D-glucose influx and efflux using rapid quenching techniques, it was demonstrated that K'" is almost 5-fold lower than that measured from time course data using integrated rate analysis (14). In addition, although the transport system displayed asymmetry at 0 and 20 "C (K'" > KOi), the Haldane requirements were satisfied (14). The inequality of Haldanes obtained using integrated rate analysis of exit was ascribed to a differential transport of a and p anomers of D-glucose (14) in spite of the recent unambiguous analysis of a- and @-D-glucose transport by both integrated and initial rate approaches that demonstrated (in 13 separate paired experiments) that no such difference existed (11).

This recent study (14) essentially confirms earlier initial rate measurements by Ginsburg et al. (26-28) using galactose (a sugar transported by the glucose transport system but with lower affinity than D-glucose). Galactose transport by red cells is asymmetric (P >> W) but displays equality in Hal- danes for Michaelis and velocity parameters for exit and entry at 10-25 "C (Kio/Koi = Vio/Voi). It should be emphasized, however, that the use of zero-trans flux data alone cannot be used to confirm or reject one- or two-site models for sugar transport. Satisfying the Haldane requirements of a passive transport mechanism does not indicate the type of mechanism but merely confirms the passive nature of the system. More sensitive tests must be employed. One such test is to determine that concentration of intracellular sugar that reduces the rate of saturated net sugar entry into cells by half (the infinite-cis entry procedure (3)) or that concentration of in-

tracellular sugar at which efflux into saturating sugar solution is half-maximal (the infinite-trans exit procedure (3)). Gins- burg and Stein (26) determined from clearly linear initial rate infinite-cis entry data and from integrated rate analyses of the time course of infinite-cis galactose entry data that the Michaelis constant for sugar efflux in the presence of saturating extracellular galactose levels was 21-25 mM. Using the zero-trans entry, exit, and exchange transport data from the same studies (26-28), the predicted Michaelis constant for efflux via the one-site carrier under infinite-trans conditions is 152 mM. Thus, although the zero-trans entry, exit, and exchange transport data were consistent with the one-site model, the results of the more sensitive infinite-&/trans procedures lead to the rejection of the one-site model.

The recent initial rate analyses (14) did not include measurements of infinite-cis or infinite-trans fluxes. Using their data, however, it is possible to calculate the predicted values for Michaelis constants for infinite-cis D-glucose influx and efflux via a one-site carrier (3). These values at 0 "C are 13 and 1 mM for infinite-cis D-ghCOSe influxlinfinite-trans exit and infinite-cis efflux, respectively, and 9 and 3 mM at 20 "C. Experimental values for D-glucose transport have been determined by rapid initial rate analysis to be 3.8 and 0.4 mM at 0 "C for infinite-trans exit and infinite-cis efflux, respectively, and 1.7 mM for infinite-cis efflux at 20 "C (see Ref. 3). Km(app) for infinite-cis entry at 20 "C, estimated by integrated rate analysis, is 2.8 mM (3). As the initial rate D-glucose and galactose transport data can be criticized neither on the basis of deviation from linearity with time nor on the basis of differential transport of a and p anomers of D-glucose, these data are sufficient to reject the one-site model. The low Km(app) site at the interior of the cell remains the major anomaly in red cell sugar transport (3).

Recent studies have suggested that the complexity of red cell D-glucose transport results from factors extrinsic to the transport mechanism. The factors could include compartmen- talization of intracellular sugar (29) and modulation of transport by intracellular ATP (9, 10, 12). In both instances, the adoption of two-site transport mechanisms was necessary to mimic the experimental data. Such transport models envisage transport proceeding via a water-filled, substrate-gated pore which, when gated by substrate at both sites of the membrane, is opened maximally (stabilized) in a configuration permitting the exchange of two sugar molecules within the channel (15).

Conclusions-Sugar-displaceable cytochalasin B binding to class I sites of the erythrocyte membrane (the sugar transporter) is systematically inconsistent with the alternating conformer (one-site) model for facilitated diffusion. The simple, two-site model is, however, consistent with the available data, suggesting that the carrier contains two substrate-bind-' ing sites (one facing the exterior of the cell and the other oriented to the interior of the cell) that exist simultaneously. In deriving the solutions to the two-site model, the assumption of rapid equilibrium kinetics was made. This assumption remains to be verified experimentally. The apparent dissociation constants for external and internal inhibitor displacement of cytochalasin B from the internal site are in substantial agreement with those for inhibition of sugar transport by external and internal inhibitor. When occupied by D-glucose (a transported sugar), the substrate-binding sites are independent. When occupied by nontransported substrate, the sites display negative cooperativity, a finding supported by recent studies of 2,N-(azidosalicoyl)-1,3-bis(mannos-4'- yloxy1)propylamide binding to the exofacial transport site in erythrocytes (30). This phenomenon presumably reflects substrate-dependent, substrate-induced conformational changes within the transporter molecule. This general view is SUP-


ported by the observed modifications of substrate affinities by tetrathionate (31) and the enhancement of fluorodinitro- benzene inhibition of sugar transport by D-glucose and pro- tection against inhibition by maltose (for review see Ref. 15). These findings provide corroborative support for recent studies of substrate-induced transporter intrinsic fluorescence quenching (9, 10) and measurements of D-glucose exchange transport (25) which indicate that the carrier contains at least two substrate-binding sites. Moreover, they provide yet further evidence to support the rejection of the one-site carrier model in favor of a two-site transport mechanism (10, 15).

1.

2.

3.

4.

5.

6. 7.

8.

9. 10. 11.

12.

REFERENCES

Kasahara, M. & Hinkle, P. C. (1977) J. Biol. Chem. 252, 7384-

Baldwin, S. A., Baldwin, J. M., Gorga, F. R. & Lienhard, G. E.

Stein, W. D. (1986) Transport and Diffusion ucross Cell Mem-

Krupka, R. M. & Deves, R. (1981) J. Biol. Chem. 256, 5410-

Gorga, F. R. & Lienhard, G. E. (1981) Biochemistry 20, 5108-

Sogin, D. C. & Hinkle, P. C. (1980) Biochemistry 19,5417-5420 Jung, C. Y. & Rampal, A. L. (1977) J. Biol. Chem. 252, 5456-

Baker, G. F., Basketer, D. A. & Widdas, W. F. (1978) J. Physiol.

Carruthers, A. (1986) J. Biol. Chem. 261, 11028-11037 Carruthers, A. (1986) Biochemistry 25,3592-3602 Carruthers, A. & Melchior, D. L. (1985) Bwchemistry 24,4244-

Hebert, D. N. & Carruthers, A. (1986) J. Biol. Chem. 261,10093-

7390

(1979) Biochim. Biophys. Acta 552,183-188

branes, pp. 231-361, Academic Press, Orlando, FL

5416

5113

5463

( L o n d . ) 278,377-388

4250

13. 14.

15.

16.

17.

18. 19.

10099 Cha, S. (1968) J. Biol. Chem. 243,820-826 Lowe, G. L. & Walmsley, A. R. (1986) Biochim. Biophys. Acta

Naftalin, R. J. & Holman, G. D. (1977) in Membrane Transport in Red Cells (Ellory, J. C., and Lew, V. L., eds) pp. 257-300, Academic Press, Orlando, FL

Jung, E. K. Y., Ching, J. J. & Jung, C. Y. (1986) J. Bwl. Chem.

Segel, I. H. (1975) Enzyme Kinetics, pp. 515-517, John Wiley &

Lin, S. & Snyder, C. E., Jr. (1977) J. Biol. Chem. 252,5464-5471 Rampal, A. L., Pinkofsky, H. B. & Jung, C. Y. (1980) Biochemistrv

857,146-154

261,9155-9160

Sons, New York

19,679-683

1908

381-397

~,~ ~. ~ ~~~

20. Gorga, F. R. & Lienhard, G. E. (1982) Bwchemistry 21, 1905-

21. Jennings, M. L. & Solomon, A. K. (1976) J. Gen. Physiol. 67,

22. Krupka, R. M. (1985) J. Membr. Biol. 83, 71-80 23. Basketter, D. A. & Widdas, W. F. (1978) J. Physiol. (Lond.) 278,

24. Wang, J., Falke, J. J. & Chan, S. I. (1986) Proc. Natl. Acud. Sci.

25. Holman, G. D., Busza, A. L. Pierce, E. J. & Rees, W. D. (1981)

26. Ginsburg, H. & Stein, W. D. (1975) Biochim. Biophys. Acta 382,

27. Ginsburg, H. & Ram, D. (1975) Biochim. Biophys. Acta 382,

28. Ginsburg, H. & Yeroushalmy, S. (1978) J. Physiol. ( L o n d . ) 282,

29. Naftalin, R. J., Smith, P. M. & Roselaar, S. E. (1985) Biochim.

30. Holman, G. D., Parkar, B. A. & Midgley, P. J. W. (1986) Biochim.

31. Krupka, R. M. (1985) J. Membr. Biol. 84, 35-43

389-401

U. S. A. 83.3277-3281

Biochim. Biophys. Acta 649,503-514

353-368

369-376

399-417

Biophys. Acta 820,235-23

Biophy~. Acta 855,115-126

Equilibrium Ligand Binding to the Human Erythrocyte Sugar ...

Documents

Transcript of Equilibrium Ligand Binding to the Human Erythrocyte Sugar ...