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THE JOURNAL OF BIOLOGICAL CHE~~IS TRYVol. 246, No. 5, Issue of March 10, pp. 1461-1467, 1971

Printed in U.S .A.

The Role of Sialic Acid in Determining the Survival ofGlycoproteins in the Circulation*

(Received for publication, November 4, 1970)ANATOL G. MORELL, GREGORY GREGORIADIS, AND I. HERBERT SCHEINBERGFrom the Department of Medicine, Albert Einstein Collegeof Medicine, New York, New York 10461JEAN HICKMAN AND GILBERT ASHWELLSFrom the National Institute of Arthritis and Metabolic Diseases,National Institutes of Health.,Bethesda, Maryland 20014

SUMMARYEvidence is presented to indicate a generalized role forthe terminal sialic acid residues of circulating glycoproteins.Upon injection into rats, all of the desialylated plasma pro-teins tested, with the exception of transfer& were promptlyremoved from the circulation and were recovered from theliver. The materials examined included orosomucoid,fetuin, ceruloplasmin, haptoglobin, arz-macroglobulin, thyro-globulin, lactoferrin, and the two gonadotropic hormones,human chorionic gonadotropin and follicle-stimulatinghormone.Competitive inhibition of hepatic uptake was demonstratedby the injection of tracer amounts of 64Cu-ceruloplasmintogether with substantive amounts of the above-mentioneddesialylated proteins or the glycopeptides derived from

them. Uptake was not inhibited by the fully sialylatedprotein or their glycopeptides.

A previous report in this series (1) documented the observa-tion that enzymatic removal of sialic acid from ceruloplasminresulted in a preparation which, upon intravenous injection intorabbits, was rapidly transferred from the circulation into theparenchymal cells of the liver. Subsequent quantitative studiesin rats (2) established that the cleavage o f only a small fractionof the sialic acid residues of ceruloplasmin was suff icient to eff ectthe removal of these molecules from the circulation.At the time of the original observation, the generality of thisphenomenon was tested with transferr in, the iron-binding proteinof normal serum. Injection of a preparation of transferrin fromwhich all of the sialic acid had been removed ensymat ically re-sulted in a serum surv ival time indistinguishable from that ofthe ful ly sialylated protein (1). As a consequence, we concluded

* This work was supported in part by Grant AM 01059 from theNational Institute of Arthritis and Metabolic Diseases of theUnited States Public Health Service, Grant G62-58 from the LifeInsurance Medical Research Fund, and a grant from the NationalGenetics Foundation, Inc.$ To whom reprint requests should be addressed.

that the striking eff ect of neuraminidase upon ceruloplasmin was,if not unique, at least not characteristic of all glycoproteins.Reinvestigation of this phenomenon, the results of which arepresented in this paper, suggests that it is transferrin which isthe exception with respect to the function of sialic acid, and thatsialic acid plays a previously unrecognized role in regulatingsurvival in the circulation of the large majority of glycoproteinstested.

MATERIALS AND METHODSClostridium perfringens neuraminidase (1.25 units per mg),

Pronase (Grade B), and galG%dase (20 units per mg) wereobtained from Worthington, Calbiochem, and General Biochem-icals, respectiveI

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1462 Role of Xialic Acid in Glycoproteins Vol. 246, No. 5

IO86

-.O... AsialatransferrinV Ceruloplasmin

Asioloceruloplasminioloceruloplasmin

MacroglobulinY

Asialoth)

Asiolohoptoglobin

\roglobulin*

Asialofetuin

11 \:...yom ;; , , , ,

0 IO 30 50 70 90MINUTES

11

-I

FIG. 1. Plasma survival time of tritiated asialoglycoproteinsin the rat. The preparation of various tritiated asialoglycopro-teins is described under Materials and Methods. Inject ions(1 ml) were made in the tail vein o f male albino rats weighingabout 200 g. Samples o f blood were taken at varying time inter-vals from the tail veins in heparinized tubes and centrifuged, andthe plasma radioactivity was determined. Animals were killed,their li ver was removed at the times indicated, and aliquots ofabout 20 mg of wet tissue were used for determination of radio-act ivi ty. 0 a. * *O, 3H-asialotransferrin, 0.3 mg, 6.6 X lo6 DPM;o- - -v , YL-ceruloplasmin, 0.3 mg; O-O, 3H-asialothyro-globulin, 0.3 mg, 12.6 X lo6 DPM. The liver (at 90 min) con-tained 23% of the radioactivity injected; O-0, 3H-asialo-czz-macroglobulin, 0.19 mg, 11.2 X lo6 dpm. The liver (at 60 min)contained 44% of the radioact ivity injected; A-A, 3H-asialo-haptoglobin, 0.12 mg, 9.5 X lo6 dpm. The liver (at 23 min) con-tained 43% of the radioact ivity injected; A-A, 3H-asialo-ceruloplasmin, 0.3 mg, 6.7 X lo6 dpm. The liver (at 14 min)contained 91% of the radioactivity injected; H--W, 3H-asialo-fetuin , 0.11 mg, 2 X lo6 dpm. The liver (at 20 min) contained55% of the radioactivity injected; O-U, 3H-asialoorosomucoid,0.13 mg, 14.4 X lo6 dpm. The liver (at 20 min) contained 98%of the radioactivity injected.Biological Company, Grand Island, New York, and a-methyl-D-galactoside was synthesized as described by Frahn and Mills (7).Crude human chorionic gonadotropin (2,270 i.u. per mg),which was obtained from Organon, Inc ., West Orange, NewJersey, was purified on either DEAE-Sephadex (8) or DEAE-cellulose (9) with subsequent isolation from carboxymethy lSephadex (10). The fina l preparation had a biological potencyin the range of 11,500 to 13,000 i.u. per mg as measured in theventral prostate weight (11) or the ascorbic acid depletion assay(12).

TANLE IE$ect of asialoorosomucoid on hepatic uptake of

E4Cu-asialoceruloplasminA tracer amount (0.3 mg) of Wu-asialoceruloplasmin wasinjected into the tail vein of male albino rats. In Experiments2 to 4, the indicated amounts of various preparations were addedto the tracer. The animals were killed after 14 min and the liver

radioact ivity was determined as described in the legend to Fig. 1.Experiment no. and addition I Amount

1. None.2. Asialotransferrin.,3. Orosomucoid . . .4. Asialoorosomucoid

w3.98.18.7

Radioactivityxovered in the liver%

9899983.6

Human pituitary follicle-stimulating hormone (LER 8690) wasthe generous gift of Dr. Grif f Ross. This material was iodinatedwith lZ51 by the method of Greenwood and Hunter (13) to yield aspecific activ ity of 35 to 75 mCi per mg. The iodinated proteinwas separated from the unreacted isotope by passage through aSephadex G-25 column and the extent of radiation damage wasascertained by the double antibody technique (14). hpproxi-mately 65 to 70% of the radioactivity was precipitated in thepresence of excess antibody. For the preparation of the asialoderivat ive, 1 mg of carrier hormone (Albert D-RA, 50 i.u. permg) was added to 1 ml of 0.05 M sodium acetate buffer, pH 5.6,in 0.15 M NaCl containing 4 X 10 cpm of 1251-FSH.1 To thiswere added 20 pg of C. perfringens neuraminidase and the samplewas incubated at 37 for 20 min. The release of sialic acid wasdetermined by the method of Warren (15). The neuraminidasewas readily removed by aff ini ty chromatography on a column ofSepharose-4B which had been coupled with rabbit antineur-aminidase antibody (16). The iodination and desialylation ofHCG were carried out in a similar fashion with essentia lly iden-tical results. The procedures for the enzymatic removal o fsialic acid from ceruloplasmin and for tritiation of the resultingasialoprotein previously reported (3) were followed for each ofthe other glycoproteins described in this report.

The glycopeptides of ceruloplasmin, transferrin, and oroso-mucoid were prepared by the method of Jamieson (17, 18).Following the separation on Sephadex G-25, the glycopeptidesolution was evaporated to dryness over NaOH and redissolvedin 0.1 M sodium acetate, pH 5.6, containing 0.17 M NaCl. Thesialic acid concentration was adjusted to that, of a 1% solutionof the protein from which the glycopeptide fraction was derived.Removal of sialic acid was accomplished by hydrolysis in 0.1 NHCl at 85 for 30 min, followed by neutralization to pH 5.6with NaOH.

RESULTSInjection of a number of tritiated asialoproteins into rats gave

rise to the pattern of survival curves illustrated in Fig. 1. Incontradistinction to the surv ival curves of native ceruloplasminand asialotransferrin, the asialo derivatives of all the otherglycoproteins disappeared promptly from the plasma. Notshown on the figure are the control curves for ~261-labeled prep-

* The abbreviations used are: FSH, follicle-stimulating hor-mone; HCG, human chorionic gonadot.ropin.

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Issue of March 10, 1971 More& Gregoriadis, Xcheinberg, Hickman, and Ashwell 1463

A4O I I I I I I I I t I I I I20 40 60 SO 100 120 140MINUTES

16C

FIG. 2. Eff ect of increasing amounts o f asialoorosomucoid uponthe circulation time of Wu-asialoceruloplasmin. Conditionswere as described in the legend to Fig. 1. All rats were injectedwith Wu-asialoceruloplasmin (0.3 mg) with or without additionof varying amounts of unlabeled asialoorosomucoid. The dashedline shows the decrease in plasma concentration of ful ly sialylatedWu-ceruloplasmin resulting from both diffus ion into extra-vascular space and normal catabolism. Additions of asialo-orosomucoid: A, none; 0,0.75 mg; l ,2.0 mg; A, 8.5 mg.arations of orosomucoid, fetuin , and art-macroglobulin, all ofwhich were indistinguishable from intact 64Cu-ceruloplasminduring the initial 60 min. In the case of 1251-thyroglobulin, thesurvival time was significantly greater than that of the asialoderivat ive, although the difference was less marked than withthe other preparations shown in Fig. 1.In every instance, the major portion of the radioact ivity lostfrom the plasma was recovered in the liver. Consequently, thecompetitive inhibition of these various desialylated proteins,with respect to hepatic uptake, was investigated. The initialexperiments revealed that when tracer amounts of j4Cu-asialo-ceruloplasmin (0.3 mg) were injected into a rat and the animalwas killed 14 min later, approximately 98% of the total radio-act ivi ty was recovered from the liver. However, when un-labeled asialoorosomucoid (8.7 mg) was injected simultaneouslywith 64Cu-asialoceruloplasmin, the amount of radioact ivity re-coverable from the liver was less than 4% (Table I). Neitherintac t orosomucoid nor asialotransferrin reduced the hepaticuptake of asialoceruloplasmin.The effect s of varying both the amount of inhibitor used andthe period of observation were next investigated and the resultsare illustrated in Fig. 2. With increasing doses of unlabeledasialoorosomucoid as the inhibitor, the survival time of thelabeled tracer rose sharply from less than 5 min to more than 2hours. Analogous experiments with other desialylated proteinsyielded similar although less striking results. When asialo-haptoglobin (10 mg) was tested as an inhibitor, the half-li fe ofthe tracer increased 8-fold to 40 min, whereas with desialylatedpreparations of crs-macroglobulin (10 mg) , thyroglobul in (20mg), and lactoferrin (5 mg), the half-life was approximatelydoubled. Comparable amounts o f the ful ly sialylated proteins

0.8OS

0 20 40 60 80 100 120 140 161M I NUTES

FIG. 3. Relationship of plasma concentration of asialoorosomu-coid to its inhibitory eff ec t. Conditions were as described in thelegend to Fig. 1. The combined injection contained Wu-asialo-ceruloplasmin (0.3 mg) and 3H-asialoorosomucoid (10 mg, 16.4 Xlo6 dpm). 0, copper-64; 0, tritium.

FIG. 4. Relationship of plasma concentration of asialofetuinto its inhibitory eff ect . Conditions were as described in the legendto Fig. 1. The combined injection contained W?u-asialocerulo-plasmin (0.3 mg) and 3H-asialofetuin (10 mg, 1.1 X lo6 dpm).0, copper-64; 0, tritium.were uniformly inef fect ive in lengthening the survival time ofthe asialoceruloplasmin.

It seemed clear from the foregoing that the amount of in-hibitor in the circulation changed constantly with time and thatthe hepatic uptake of the tracer occurred only after a criticalserum concentration of the inhibitor was reached. In order todefine this relationship more precisely, a series of experimentswas undertaken wherein both inhibitor and asialoceruloplasmintracer were labeled with tritium and copper-64, respective ly.

The survival curves resulting from the simultaneous injectionof 3H-asialoorosomucoid (10 mg) and 64Cu-asialoceruloplasmin(0.3 mg) are shown in Fig. 3. For approximately 2 hours, thelatter remained viable in the circulat ion, during which time theconcentration of the former decreased stead ily. As the plasma

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1464 Role of Xialic Acid in Glycoproteins Vol. 246, No. 5

I I I I ! I I 1 B I I0 20 40 60 00 100 120 140MINUTES

FIG. 5. Relationship of plasma concentration of asialohapto-globin to its inhibitory eff ect . Conditions were as described inthe legend to Fig. 1. The combined injection contained 64Cu-asialoceruloplasmin (0.3 mg) and 3H-asialohaptoglobin (10 mg,9.2 X lo6 dpm). 0, copper-64; 0, tritium.

o.zo I 50 100 150 200MINUTES

FIG. 6. Concentration-dependent clearance of 64Cu-asialo-ceruloplasmin from the plasma. Conditions were as described inthe legend to Fig. 1. The radioactivities of 20-J aliquots ofplasma, at each time point, were expressed as a fraction of theradioact ivity present in the firs t sample (3 min) multiplied by thedose injected (milligrams) and plotted on the ordinate as milli-grams of asialoceruloplasmin present in plasma. In the upper-most curve, 38 mg of Wu-asialoceruloplasmin were injected; inthe lower three curves, 15.5, 8.5, and 4.7 mg, respectively , wereinjected.concentration of the tritiated inhibitor approached 1% of theinjected dose per ml, the slope of both survival curves increasedsharply and both proteins disappeared rapidly from the circula-tion. A similar pattern of behavior was seen when tritiatedasialofetuin was used as the inhibitor (Fig. 4). In the case ofhaptoglobin, the tracer preparation disappeared at a constant,

0 5 15 25 35 45 55 65 75MINUTESFIG. 7. Inhibitory ef fec t of asialoglycopeptides on the clearance

of asialoceruloplasmin. Glycopeptides from ceruloplasmin andorosomucoid were isolated and desialylated as described underMaterials and Methods. Other conditions were as describedin the legend to Fig. 1. 0, 64Cu-asialoceruloplasmin (0.44 fig )plus the sialylated glycopeptides derived from 21 mg of cerulo-plasmin; IJ, Wu-asialoceruloplasmin (0.21 mg) plus the sialylatedglycopeptides derived from 10 mg of orosomucoid; l , Wk-asialo-ceruloplasmin (0.44 mg) plus the desialylated glycopeptidesderived from 21 mg of asialoceruloplasmin; n , 64Cu-asialocerulo-plasmin (0.21 mg) plus the desialylated glycopeptides derivedfrom 10 mg of orosomucoid.although markedly diminished, rate throughout the entire 2-hourperiod (Fig. 5).A more detailed study o f survival time as a function of dosageis shown in Fig. 6. The half-life of injected 64Cu-asialocerulo-plasmin was neither constant nor proportional to the dosage.Instead, it decreased intermittently throughout the experimentas a function of the changing plasma concentration rather thanof the amount injected.As a consequence of the demonstration that several desial-ylated glycoproteins acted effect ive ly as inhibitors of hepaticuptake, it appeared unlikely that specific conformational modi-fication was responsible for the inhibitory act ivi ty. In order toexclude this possibility and to define more precisely the natureof the inhibitor, the intact glycoprotein was destroyed by treat-ment with Pronase and the resulting glycopeptide moiety wasrecovered. Upon injection into rats, the glycopeptides isolatedfrom ceruloplasmin and from orosomucoid had no eff ect on thesurvival of the labeled tracer in the circulation. However, upondesialylation, the glycopeptides from both proteins were effect ivein inhibiting uptake of asialoceruloplasmin by the liver (Fig. 7).In clear contrast, neither the intact nor the asialoglycopeptidesisolated from transferrin inhibited the rapid accumulation oftracer in the liver (Table II). Similarly, free galactose, CFmethyl-n-galactoside (30 mg), and lactose (30 mg) were ineffec--tive as inhibiting agents.

With the realization that the viabi lity of many ( if not most)of the serum glycoproteins depends upon the presence o f theirnormal complement of sialic acid residues, consideration wasgiven to another category of biologically potent glycoproteins,i.e. the gonadotropic hormones. The biological act ivi ty of two,members of this group? human chorionic gonadotropin and__

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Issue of March 10, 1971 Morell, Gregoriadis, &heir&erg, Hickman, and Ashwell 1465TABLE II

Comparison of inhibitory effectiveness of glycopeptides isolatedfrom fransjerrin and orosomucoid

A tracer amount (0.2 mg) of 64Cu-asialoceruloplasmin wasinjected into the tail vein of male albino rats. In Experiments 2to 5, the indicated amounts of various preparations were added tothe tracer. All plasma samples were collected 15 min after in-jection and the radioactivity determined as described in thelegend to Fig. 1.Experiment no. and source of glycopeptide

1. None........................2. Native transferrin .3. Desia lylated transferrin.. .4. Native orosom ucoid.. . . .5. Desialylated orosomucoid . .

s-.

Amountsmg30301010

Radioactivityremaining incirculation% dose/ml plasma

0.60.10.20.59.8

The weight of glycoprotein from which the added glycopep-tides were derived.

.f ;\ , ,I.0 5 IO 20 30

MINUTESFIG. 8. Plasma survival time of native and desialylated human

chorionic gonadotropin in the rat. The preparation of HCGwas purified, labeled with Y, and desialylated as described underMaterials and Methods. Each injection (1.0 ml) contained2.0 to 2.6 X 10 cpm, of which 90% was precipitable with specificantisera. Injections were made in the tail vein of Sprague-Dawley female rats weighing about 200 g. At the indicated timeintervals, blood was collected in heparinized tubes and centri-fuged, and the residual radioactivity was determined on an aliquotof the plasma. The same aliquot was then assayed by the doubleantibody technique and all values were corrected for immuneprecipitable counts. l , 1261-HCG; A, W-asialo-HCG plus 5mg of asialoorosomucoid; H, 1261-asialo-HCG. The slope of thiscurve was unaffected by the addition of 5 mg of fully sialylatedorosomucoid. In either case, the liver contained 77yo of therecoverable radioact ivity at the end of the experiment (30 mm),in the lowermost curve.follicle-stimulating hormone, is known to be destroyed uponexposure to neuraminidase (19). Since the standard hormoneassay involves injection of the active principle into an appro-priately sensitized animal, we thought it possible that the po-

i tI I I I I

1 5 IO 20 30MINUTESFIG. 9. Plasma survival time of native and desialylated follicle-

stimulating hormone in the rat. The conditions of this experi-ment were similar to those described in the legend to Fig. 8 withthe exception that 3.0 to 3.6 X 10 cpm of W-FSH were injected,of which approximately 70yo was precipitable by specif ic antisera.0, W-FSH; A, W-asialo-FSH plus 5 mg of asialoorosomucoid;n , lz51-asialo-FSH. The slope of this curve was unaffected by theaddition of 5 mg of ful ly sialylated orosomucoid. In either case,the liver contained 72% of the recoverable radioactivity at theend of the experiment (30 min), in the lowermost curve.tency lossof thedesialylated preparationsmight be correlated withtheir rapid removal f rom the circulation and consequent de-struction by the liver. By analogy with the behavior of theserum glycoproteins, we also anticipated that the rapidity oftheir clearance from the circulation might be diminished by thesimultaneous injection of an appropriately desialylated inhibitoryagent.

In order to test this hypothesis, highly potent preparations ofHCG and FSH were desialylated enzymatically with C. perfrin-gens neuraminidase. After removal of the neuraminidase byaff ini ty chromatography, the hormones were reisolated andassayed for both biological act ivi ty and immunoprecipitability.As expected, the treated preparations were biologically inert,although their abil ity to react with specif ic antisera was unim-paired.Upon injection into rats , the lz51-labeled, desialylated hor-mones disappeared rapidly from the circulation with the majorportion of the total radioact ivity being recoverable from the liver(Figs. 8 and 9). The close parellelism of these results with thoseof the serum glycoproteins was underlined by the marked in-crease in surviva l time in the presence of asialoorosomucoid;intact orosomucoid was without eff ect .

DQferentiation from Phagocytosis-The rapid clearance ofneuraminidase-treated glycoproteins and their resultant localiza-tion in the liver is reminiscent of the activit y of the systemicreticuloendothelial cells in clearing the circulation of foreignparticles. The ability of these cells to phagocytize a variety ofsuch materials as bacteria, carbon suspensions, and denaturedproteins has been correlated with their role in cellular defense.No such mechanism is apparent in the highly selective removal of

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1466 Role oj Xialic Acid in Glycoproteins Vol. 246, No. 5Tanm III

Inhibitor specificity for hepu tic uptake by Kupffercells and hepatocytes

In Experiments 1 and 2, *311-heat-dellatured albumin (0.5 mg)was injected and in Experiments 3, 4, and 5 Wu-asialocerulo-plasmin (0.3 mg) was injected. The animals were killed 10 minafter injection and the liver radioactivity determined as de-scribed in the legend to Fig. 1.Experim ent no. and radioactive traceused

1. I-1~eat-denatured albu-min2. 311.Ileat-denatured nlbu-min3. 64Cu-~~laialoceruloplasn~in4. Wu-Asialoceruloplasmin

5. 6*Cu-Asi,zloeeruloplasmin

r j Inhibitor added AIlUXln

NoneAsialoorosomucoid I 10NoneNoniodinuted heat-t 20denatured albuminAsialoorosomucoid 10

Radio-ictivitgreiov-ered inliver

even l)art,ially desinlylatcd glycoproteins (a). The differencebetween these two phenomena was implied in an earlier studywherein the hepatic uptake of 3H-asialoceruloplasmin was moni-tored 1,~ serial historadioautograms. -211 of the radioactivity wasseen to be prcscnt in the parenchymal cells; none ~1-a~ observedin the I- l)e their nor~n:tl catabolic paths-a>-. In the case of thegonadot,ropic Irormoncs, theae findings proride :I, reasonableesplannt,ion for the well documented observation Ihat remova,of sialic acid is invariably accompanied by extensive loss ofhormonal acti vit ,y (LS).

Further differentiation betIT-een the t\T-o systems was obtainedby comparison of t,he quantitatire aspect,s of clearance. De-tailed studies 011 the kinetic parameters of phagocytosis indicatedthe surv ival time of phagocytized colloids was shown to beproportional to the dose :tdministercd nald to be constant through.-out the duration of the esperirncnt (20). In contradistinction,the surv ival time of aaialoceruloplasmin was not proport,ionalto the dose inject,ed nor was it constant throughout the esperi-ment (Fir?;. 6). In short, the nrailuble evidence points t ,o theidentification of the hepatocytc, and not the Kupffe r ~11, as thesit,e of active :rccumulation of nsi~tloglycoproteiiis.

It the present timc, little information is available as to themechanism of bintling and trmlsport of desialylated proteins intohepatocytcs. The number of binding sites appears, ho\\-ever, tobe limit,ed since the>- are readily saturated by the injection ofmilligram quantities of an appropriate nsialoprotein. Thesubsequent intracellular ljrotcin transport and lysosom~l cntabo-lism (21) would t,hcn serve t,o make such sites availahlr forfurther ul)tnkc.

The curiousl>r ticlccti~e nature of these receptor sites wasrerealed by the Ironase digest,ion exlpcrimcnts, which shoTI-edthat the requirements fol , binding do not include a conformation-ally intact protein but are satisfied by the asialoglycopeptidesfrom many llrotciiis (Fig. 6) with the exception of trunsferrin(Table II) . Xe\-crlheless, the composition of the two carbohy-dratc chains of tmnsferrin does not appear to be strikinglydissimilar to that of the other glycoproteins examined (18).

These results, and the lack of inhibition by simple carbohydrateanalogues, suggest :I spec ific ity a,nd complexity of the glJ-copep-tide structure which rcm:tin to be elucidated.

-1 secoud point of importance to emerge from these studies is Acknowledgments-.Tlle authors are h ~rat~eful to Drs. G. Rossthe recoguit,iorr of the principle of competitive hcpatic inhibiiion. and J. Vaitukaitis for their inT-nluable help in assaying the bio-

Upon the simultaneous injection of two asialoproteins of varyingsusceptibi lity for clearance, a preferential removal of one is seen.A good example of this variable binding behavior is illustratedin Fig. 3. In the presence of excess asialoorosomucoid, there is acomplete exclusion of asialoceruloplasmin from the liver. At thepoint at which the plasma concentration of the former fa lls to acritical low level, both proteins disappear rapidly and at approxi-mately t,he same rate. .Gnlohaptoglobin, on the other hand,exhibits a binding eff iciency for the liver similar to that of asialo-ceruloplasmin, since the latter protein disappears from the plasmacontinuously and at a constant rate in the presence of excessinhibitor (Fig. 5).

With the development of the inhibition technique, it becamepossible to re-examine the role of the Kupff er cells in the selectivehepatic uptake of asialoglycoproteins and to confirm the earlierhistoradioautographic evidence for the participation of paren-chymal cells in this phenomenon (I). As expected, a directchallenge to the reticuloendothelial system of the rat with heat-denatured 1311-albumin resulted, within 10 min, in an incorpora-tion of 6374, of the injected dose in the liver. The hepatic ac-cumulation of the denatured albumin was completely unaffectedby asialoproteins. Conversely, the simultaneous injection o fheat-denatured albumin, a potent blocking agent for Kupffer celluptake, was without eff ect upon the survival of asialoceruloplas-min (Table III). Thcsc results are inconsistent with t,he deposi-tion of the asialoglycoproteins in the Kupffer cells and provideadditional supportive evidence that the parcnchymal cells arethe locus of accumulation in the liver.

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Issue of March 10, 1971 More& Gregoriudis, Scheinberg, Hickman, and Ashwell 1467logical act ivi ty and immunocompetence of the gonadotropic 10.hormones and to Julie Windsor for skillfu l technical assistance. 11

REFERENCES 12:1. MORELL, A. G., IRVINE, R. A., STERNLIEB, I., SCHEINBERG,I. H., AND ASHWELL, G., J. Viol. Chem., 243, 155 (1968).2. VAN DEN HAMER, C. J. A., MORELL, A. G., SCHEINBERG,

I. H., HICKMAN, J., AND ASHWELL, G., J. Biol. Chem., 246,4397 (1970). 3. MORELL. A. G.. VAN DEN HAMER. C. J. A.. SCHEINBERG. I.H., A& A.SH&LL, G., J. Biol. kern., 241: 3745 (1966). 4. WHITEHEAD, P. H., AND SA~~MONS, H. G., Biochim. Biophys.Ada, 124, 209 (1966).5. SMITH. H.. EDMAN. P.. AND OWEN. J. A.. Nature, 193, 286(196i). 6. CONNELL, G. E., AND SHAH, R. W., Can. J. Biochem. Physiol.,39, 1013 (1961).7. FRAHN, J. L., ANDMILLS, J. A., Aust. J. Chem., l&l303 (1965).8. BAHL, 0. P., J. Biol. Chem., 244, 567 (1969).9. REISFELD, R. A., AND HERTZ, R., Biochim. Biophys. Acta, 43,540 (1960).

13.14.15. WARREN, L., J: Biol. C hem., 234, 1971 (1959).16. VAN HALL. E. V.. VAITUKAITIS. J.. Ross. G. T.. HICKMAN.17.18.19.20.

21.

VAN HELL, H., MATHIJSSEN, R., AND HOMAN, J. D. H., ActaEndocrinol., 69, 89 (1968).MCARTHUR, J. W., Endocrinology, 60, 304 (1952).PARLOW, A. F., in A. ALBERT (Editor), Human pituitarygonadotropins, Charles C Thomas Publisher, Springfield,Illinois, 1961, p. 300.GREENWOOD, F. C., AND HUNTER, W. M., Biochem. J., 89,114 (1963).ODELL, W. P., Ross, G. T., AND RAYFORD, R. L., J. C&z. In-vest, 46, 248 (1967).

J., AND ~SHWE~L, G., Endocr&olbgy, 88; 456 (1971). .JAMIESON, G. A., J. Biol. Chem., 240, 2019 (1965).JAMIESON, G. A., J. Biol. Chem., 240, 2914 (1965).MORI, K. F., Endocrinology, 86, 330 (1969).BENACERRAF, B., in R. W. BRAUER (Editor), Proceedings of

the Symposium on Liver Function, American Institute ofBiological Sciences, Washington, 1958, p. 205.GREGORIADIS, G., MORELL, A. G., STERNLIE B, I., AND SCHEIN-

BERG, I. H., J. Biol. Chem., 246, 5833 (1970).

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Morell AG, 1971

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