THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 249, No. 24, Issue of December 25, PP. 7848-7856, 1974

Printed in U.S.A.

Studies on Receptor-mediated Activation of Adenylyl Cyclases

I. PREPARATION AND DESCRIPTION OF GENERAL PROPERTIES OF AN ADENYLYL CYCLASE

SYSTEM IN BEEF RENAL MEDULLARY MEMBRANES SENSITIVE TO NEUROHYPOPHYSEAL

HORMONES*

(Received for publication, December 20, 1973)

LUTZ BIRNBAUMER AND PO-CHANG YANG

From the Department of Physiology, Northwestern

SUMMARY

With use of a discontinuous sucrose density gradient centrifugation in the presence of 1 mM EDTA and 10 mM Trls-HCl, pH 7.5, membrane particles were prepared from beef renal medulla that are 4- to B-fold enriched in fluoride- sensitive and 8- to 12-fold enriched in neurohypophyseal hormone (NHH)-sensitive adenylyl cyclase activity. Aver- age yield of the NHH-sensitive adenylyl cyclase activity was 30 to 35% ; average yield of membrane protein, starting with 80 g of beef renal medulla, was 200 to 250 mg. These mem- branes also contain a prostaglandin-sensitive adenylyl cyclase system, the activity of which is additive to that sensi- tive to NHH.

Arginine-vasopressin, lysine-vasopressin, and oxytocin stimulate enzyme activity half-maximally at 10m9, 10p9, and 10m7 M, respectively. The response of the renal medullary adenylyl cyclase activity to NHH is extremely dependent on the concentrations of magnesium and ATP used in the assay. Response is maximal at low concentrations of magnesium and at concentrations of ATP varying from 0.07 to 0.10 mrd. Increasing the concentration of magnesium results in selec- tive stimulation of basal activity with concomitant loss of the relative stimulation by NHH. Increasing or decreasing the concentration of ATP results in selective loss of activity due to NHH stimulation, also leading to reduced relative stimu- lation by NHH. GTP at 10e5 M inhibits stimulation of the beef renal medullary adenylyl cyclase by NHH.

5’-Adenylyl-imidodiphosphate (AMP-PNP), an analogue of ATP that is not hydrolyzed by membrane ATPases, was evaluated as substrate for renal adenylyl cyclase. Depend- ing on the concentration of the analogue and magnesium used, AMP-PNP yields activities that are between 10 and 25 % of those obtained with ATP. Maximal hormonal stimu- lation requires higher concentrations of AMP-PNP than ATP; excess AMP-PNP does not result in inhibition of hormonal stimulation. Activities with the analogue exhibit higher pH optima than with ATP. The possibility that these differ- ences in activity are due to a different exit rate of inorganic imidodiphosphate as compared to PPi or to the different

* This research was supported in part by Grants HD-06273 and HD-06513 from the United States Public Health Service. We are unable to honor reprint requests.

University Medical Center, Chicago, Illinois 60611

divalent cation binding properties of AMP-PNP and ATP, or both, is discussed.

The extreme sensitivity of hormonal stimulation to varia- tions of nucleotide concentration, be it ATP or GTP, coupled to the relatively high yield of activity from beef renal medulla, make this preparation useful for the study of modes of regula- tion of hormonal stimulation of adenylyl cyclase systems.

In addition, several minor modifications of the procedure of Krishna et al. (KRISHNA, G., WEISS, B., AND BRODIE, B. B. (1968) J. Exp. Ther. Pharmacol. 163, 379-385) for isolation and determination of cyclic adenosine 3’: 5’-monophosphate (CAMP) formed during incubations for adenylyl cyclase are described. They result in higher yields of CAMP (60 to 65 %) and in lower reaction blanks (0.0005 to 0.0010% of initially added ATP).

Adenylyl cyclases (ATP : pyrophosphate-lyase (cyclizing), EC 4.6.1.1) are important models for the elucidation of molecular events involved in initiation of hormone action and have there- fore received much attention during recent years. Studies with fat cells have revealed that these enzyme systems are multi- molecular complexes asymmetrically arranged within the plasma membrane and have led to the clear recognition of at least two functional components: (a) a hormone receptor, proteic in nature and located on the outer surface of the membrane; and (b) a catalytic unit exposed at the inner side of the membrane and accessible to its substrates (I-3). In addition, studies with rat liver adenylyl cylase revealed that (a) both the catalysis of ATP to CAMP’ by enzyme and the translation of receptor- hormone interaction into activation of the catalytic unit are dependent on membrane lipids (4, 5) ; (b) the interaction of hormone with its receptor is rapidly reversible (6); (c) the process or processes coupling hormone receptor to catalytic unit require a guanyl nucleotide for proper operation (7) ; and (d) both the hormone receptor and the enzyme exist in rapidly reversible

1 The abbreviations used are: CAMP, cyclic adenosine 3’:5’- monoohosnhate: AMP-PNP. 5’-adenvlvl-imidodinhosphate; NHH: ne&ohypophyseal hormone; PC-E;, prostagiandk El: PNPi, inorganic imidodiphosphate.

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states of activity (6). Finally, studies with adenylyl cyclases from cat heart and adrenal cortex have revealed that certain phospholipids and divalent cations may also play specific roles in regulating hormonal stimulation (8, 9). For a recent general review of the subject see Perkins (10).

Despite all of these studies, the information gained is indirect and it has not yet been possible to isolate and characterize any of the molecular components comprising these complex mem- brane-bound systems. Precise information as to the nature of the coupling process(es) connecting hormone receptor to catalytic unit is missing.

Neurohypophyseal hormone (NHH)-sensitive adenylyl cyclase systems may be appropriate to gain insight into the processes that regulate the hormone receptor-dependent activation and perhaps, to isolate and characterize one or more of its compo- nents. Chase and Aurbach (11) demonstrated the existence, in renal medullary homogenates, of a NHH-sensitive adenylyl cyclase system which was later shown to be stable to freezing and which could be amenable to partial purification so as to initiate isolation of components. One very desirable feature of NHH-sensitive systems is that the chemical characteristics of the hormones activating them are well known and that it is feasible to synthesize and derivatize them so as to isolate a receptor for NHH by affinity chromatography or affinity labeling techniques, or both (12-15). The recent description by Walter et al. (16) and Von Dreele et al. (17) of the conformation of [Lys*]vasopressin in solution should be of great help in designing appropriate derivatives.

The purpose of this paper, the first in a series dealing with the mode by which hormone receptors affect adenylyl cyclases, is to describe a method for the preparation of membrane particles from beef renal medulla containing a highly sensitive, highly responsive, lo-fold purified NHH-sensitive adenylyl cyclase system, and to present its general characteristics with respect to hormone and substrate dependence. In addition, AMP-PNP, an analogue of ATP not hydrolyzed by ATPases (7, 18), was evaluated as a possible substitute for ATP, the natural substrate for the adenylyl cyclase reaction. It was found that maximal activities obtained with the analogue are significantly less than those obtained with ATP and exhibit destinct pH optima.

EXPERIMENTAL PROCEDURES

Materials

Synthetic [Ar$]vasopressin (450 units per mg) and [Lys8]vaso- nressin (270 units ner ma) were a generous gift from Dr. R. Walter, Department of Physioggy, Mount Sinai-School of Medicine of the City University of New York, New York, N. Y. Synthetic oxytocin (500 units per mg) was a generous gift from Dr. G. Flouret, Department of Physiology, Northwestern University School of Medicine, Chicago, Ill. PGE, was a generous gift from Dr. J. Pike. The Uniohn Co.. Kalamazoo, Mich. Analvtical grade (AG) ‘Dowex ,%-X4 was ‘purchased from Bio-Rad Labo- ratories, Richmond, Calif. Unlabeled 5’-adenylyl-imidodiphos- phate was purchased from International Chemical and Nuclear Corp., Irvine, Calif. ATP (Tris salt), CAMP, GTP, Buffer A (2,2-bis(hydroxymethyl)-2,2’,2”-nitriloethanol-propane) Sigma Chemical Co., St. Louis, MO.; and creatine phosphate and creatine kinase were from Calbiochem, La Jolla, Calif. The following liquid scintillation mixtures were used: Aquasol from New Eng- land Nuclear, Worcester, Mass.; PCS from Amersham-Searle, Arlington Heights, Ill.; and 3a-70B from Research Products Incorporated (RPI), Elk Grove Village, Ill.

Radiochemicals

[8H]cAMP was purchased from New England Nuclear. [c+P]- ATP (1.5 to 7.0 Ci per pmole) and [&2P]AMP-PNP were purchased

from International Chemical and Nuclear Company, Irvine, Calif. Labeled AMP-PNP was purified from an unknown radio- impurity by applying a 4-ml sample to a column (0.9 X 19 cm) of Dowex 5OW-X4 (200 to 400 mesh, H+ form) and eluting with HzO. Eighty per cent of the applied [a-52P]AMP-PNP was recovered in the 5th throuah 9th ml of eluate. The radioimpuritv (that in- creased with the age of the batch of labeled AMP-PNP ‘and be- haved like CAMP in two chromatographic systems: polyethyl- eneimine cellulose thin layer chromatography developed with 1 M LiCl and Dowex 5OW-X4 column chromatography (200 to 400 mesh, H+ form, 0.6-cm inside diameter, 1.5-ml resin bed) using Hz0 as the eluent) remained on the column. The purified ma- terial (acid form of AMP-PNP) was made 5 mM with respect to Tris base, stored frozen at -2O”, and used within 10 days of its reported synthesis.

All other reagents were commercial preparations of the highest purity available and used without further purification.

Preparation of Beef Renal Medullary Membranes Containing NHH-stimulated Adenylyl Cyclase Activity

Beef kidneys were obtained fresh from the slaughterhouse, placed in ice-cold 0.25 M sucrose containing 1.0 mM EDTA, and transported to the laboratory. Medullary sections were excised within 2 hours of killing the animal. All subsequent steps were carried out at 4”. Routinely, between 80 and 90 g of renal medulla were obtained from two beef kidneys, each weighing approxi- mately 1.0 kg. Homogenization was then initiated by subjecting 80 g of renal medulla in a Sorvall Omni-Mixer to five 2-s bursts at full speed in the presence of 160 ml of 1.0 mM KHC03. The re- sulting mixture was filtered through two layers of gauze to sepa- rate connective tissue; the residue on the gauze was washed once with 40 ml of 1.0 mM KHCOa, and the washing fluid was collected. The combined filtrate and washing fluid were then further homoge- nized in a Dounce homogenizer (Blaessig Glass Co,, Catalog No. 518) by subjecting 40.ml aliquots to 5 strokes with the loose pestle. The resulting suspension (about 250 ml) was diluted 7-fold with ice-cold 1.0 mM KHC03, stirred for 3 min, and sequentially filtered through two and four layers of gauze as described in Steps 1 through 8 of Neville’s technique for preparation of liver plasma membranes (19). This fraction was called the (‘crude homoge- nate.” It was centrifuged for 5 min at 160 X g in a Sorvall RC-3 refrigerated centrifuge. The pellet (P160) contained unhomoge- nized tissue fragments, nuclei, and major cell debris (as observed under the phase microscope), as well as 40yo of the increase in adenylyl cyclase activity due to [Argslvasopressin addition (Table I). The supernatant fraction (&co) was centrifuged further for 30 min at 1,500 x g, yielding a pellet (P160-1,500) containing most of the adenylyl cyclase activity present in S160 and a supernatant fluid (SIBO-l,SOO) containing less than 7% of the adenylyl cyclase activitv. Membranes present in Plsa.l. 600 containing NHH- sensitive adenylyl cyclase activity were further purified by dis- continuous sucrose density gradient fractionation using a SW 27 rotor in the L2-65B Beckman preparative ultracentrifuge. The centrifuge tubes (40-ml capacity) were filled with (from bottom to top): 10 ml of 41.5% sucrose, 7.5 ml of 37.Oyc sucrose, 5 ml of 31.0yo sucrose, and 15 ml of P160-1.5~~ containing, in 1 mM KHCO,, particles derived from 120 ml of crude homogenate, i.e. from about 6.5 g of renal medulla. All sucrose solutions were made with a solution of 1.0 mM EDTA and 10 mM Tris-HCl, pH 7.5. Sucrose concentrations are expressed in per cent (w/w) and were adjusted with the aid of an Abbe refractometer at room temperature to within 0.2%. Centrifugation was for 2 hours at 27,000 rpm and 5”. Membranes at the various interfaces were collected with the aid of a cold 5.0.ml syringe fitted with a 4-inch long No. 18 stainless steel needle. Membranes containing highest NHH-sensitive adenylyl cyclase activity were collected at the interface between 37.0 and 41.5’% sucrose. They were fractionated in 0.5-ml aliquots and kept frozen at -70” until used. This membrane fraction will be called throughout “renal medullary membranes.”

Adenylyl Cyclase Assay

Unless otherwise stated, fractions to be tested for their adenvlvl cyclase activity (30 to 80 ig of membrane protein) were incubated in a final volume of 0.05 ml containine 1.0 mM ~o~-~~P]ATP (100 to 200 cpm per pmole), 5.0 mM MgCl*, l.imM EDNA, 1.6 mM cAMP, 20 mM creatine phosphate, 0.2 mg per ml of creatine kinase (20 to

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30 units per mg), and 25 mrvr Buffer B (2,2-bis(hydroxymethyl)- 2,2’,2”-nitriloethanol-propane-HCl) pH 8.0. Incubations were for 10 min at 37” and were terminated by the addition of 0.1 ml of “stopping solution,” composed of 10 mM [3H]cAMP (approxi- mately 10,000 cpm), 40 mM ATP, and 1.0% sodium dodecyl sulfate, followed by immediate boiling for 3 min.

Isolation of CAMP

The [32P]cAMP formed was isolated by a slight modification of the method of Krishna et al. (20) that yields better reproducibility between replicates and lower reaction blanks. Hz0 (1.0 ml) was added to the incubated samples which were then transferred with Pasteur pipettes to Dowex 50 columns (0.6.cm inside diameter, 1.5 ml of packed Bio-Rad AG 50-X4, 200 to 400 mesh, H+ form) that have been subjected to at least three “regeneration cycles” (see below). The columns were then sequentially washed with 1.0, 3.0, and 3.0 ml of HzO. The last 3.0 ml, containing approxi- mately 80% of the applied CAMP, were collected and 0.2 ml each of 0.15 M Ba(OH)z and about 4yo ZnSOd was added in that se- quence The resulting precipitates were removed by filtering the suspensions through BaS04 filters (see below). The BaS04 filters were then washed once with 1.0 ml of HzO. The filtrates (pH 9.0 to 9.5) and the washing fluids were directly collected in counting vials containing 15 ml of Aquasol or 11 ml of PCS or 3a-70B.

Regeneration of Dowex 60 Columns-This process was carried out three times before the columns were used for the first time and once between each use in the adenylyl cylase assay. One re- generation cycle is formed by successive washing of the column packed with 1.5 ml of Bio-Rad AC 5OW-X4 (200 to 400 mesh) with 20 ml of HzO, 20 ml of 2 N NaOH, 20 ml of’HtO,20 ml of 2 N HCl, and 3 X 20 ml of HzO. Using this regime a single Dowex column has been used in up to 100 separate adenylyl cyclase assays yield- ing good recovery of CAMP without noticeable increase of the reaction blank of the assay.

BaSOd Filters-These are prepared by simultaneous addition of 0.4 ml each of 0.15 M Ba(OH)z and 4$& ZnSOb to tight glass-wool plugs placed in the tip of glass columns (0.6 X 15 cm), followed by a single washing step with 20 ml of HtO.

The above methodology differs from that previously described (21, 22) in that (a) sodium dodecyl sulfate was included in the stopping solution, (5) Dowex 5OW-X4 (200 to 400 mesh) was substi- tuted for Dowex 5OW-X8 (100 to 200 mesh), (c) the volume of resin bed was increased from 1.0 to 1.5 ml, (d) the resin was exten- sively washed with alternating solutions of NaOH and HCl before use, and (e) the radioactive mixture of BaS04 and Zn(OH)* formed during the negative adsorption step was removed by a more effi- cient filtration method rather than by centrifugation. Over-all recovery of CAMP was between 60 and 65%. Significantly lower reaction blanks were obtained under these conditions, ranging, in the presence of up to 150 rg of membranes protein, from 0.0005 to 0.0010% of the initially added ATP. Replication of duplicate samples was within 5% of the mean under all conditions of assay, except when basal activity was determined at times shorter than 2 min. Under these conditions the counts per min of ATP con- verted to CAMP were equal to or less than the reaction blank and reproducibility of the assay was reduced to within 10% of the mean.

To decrease reaction blanks in experiments with AMP-PNP, the final boiling step was omitted. The sodium dodecyl sulfate- containing stopping solution alone was sufficient to stop the reactions effectively. Under these conditions, the reaction blanks with the synthetic nucleotide were between 0.0008 and 0.0025ye of the initially added [(u-32P]AMP-PNP and reproducibility was within 10% of the mean.

Expression of Results

Adenylyl cyclase activity in figures and tables refers to the conversion of both substrates of adenylyl cyclase, 5’-adenylyl- pyrophosphate (ATP) or 5’.adenylyl-imidodiphosphate (AMP- PNP) to CAMP and PPi (or PNPi) expressed, unless otherwise indicated, in nanomoles of CAMP formed per 10 min per mg of membrane protein.

Marker Enzymes

5’-Nucleotidase (EC 3.1.3.5) was measured by the method of Bodansky and Schwartz (23) and glucose 6-phosphatase (EC

3.1.3.9) by the method of Swanson (24), with the addition of 1 mM EDTA. These enzymes were assayed in 0.1 ml of appro- priate medium and incubations were at 30”. Incubation time varied between 10 and 90 min depending on enzyme fraction and activity measured. Activities were proportional to enzyme concentration. Reactions were stopped by addition of 0.025 ml of 50% trichloroacetic acid. The precipitate formed was removed by centrifugation and the liberated Pi was determined on 0.05.ml aliquots by the method of Fiske and SubbaRow (25). Succinate- cytochrome c reductase was measured by the method described by Fleischer and Fleischer (26).

Protein was measured by the Lowry procedure (27) using bovine serum albumin as standard.

RESULTS

Preliminary experiments indicated that stimulation of adenylyl

cyclase by [Arg8]vasopressin in crude homogenates of beef renal

medulla, as well as in low speed particulate fractions, varied from as little as 20 y. up to 200 %, depending on the kidney used.

Mean stimulation was approximately 80%. Fractionation on a continuous sucrose gradient showed that low speed particles

contain in addition to [Arg*]vasopressin-stimulated adenylyl cyclase system other adenylyl cyclase activities not responsive

to the neurohypophyseal hormone (Fig. 1). Based on the data obtained from the continuous sucrose gradient we designed the purification scheme described under “Experimental Procedures”

and used a discontinuous gradient formed by (from bottom to

top of tube) 41.5, 37.0, and 32.5oj, sucrose in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA. As can be seen in Table I membranes collected at the interface between 37.0 and 41.5 y. sucrose showed

maximal [Arg*]vasopressin responsiveness. Purification of ac- tivity due to [Arg8]vasopressin addition (last two columns of

Table I) was in the order of 8- to Il-fold, with a yield ranging

between 30 and 35%. Freezing resulted in approximately 20 to

30% loss of all expressions of adenylyl cyclase activity (basal,

I.0 r

O-

,I.00

FRACTION NUMBER (0.5 ml each )

FIG. 1. Sucrose density gradient profile of adenylyl cyclase activities present in homogenate of beef renal medulla. Crude homogenate (15 ml) was centrifuged for 30 min at 1,500 X g and 4”. The resulting pellet (10.3 mg of protein) was suspended in 1.5 ml of 10 mM Tris-HCl, pH 7.5-l mM EDTA and layered on a 9.5.ml 28 to 52yo (w/w) sucrose gradient prepared in 10 mM Tris- HCl pH 7.5-l mM EDTA. Centrifugation was for 16 hours at 40,000 rpm in a Beckman SW 40 rotor and at 4”. After centrifuga- tion, the gradient was lifted out using an ISCO model 183 density gradient fractionator and fractionated in 0.5.ml aliquots. Frac- tions were analyzed for their content of basal (0-O ), [Arg8]- vasopressin-stimulated (O--O ), and fluoride-stimulated (A---A), adenylyl cyclase activities, protein, and sucrose con- centration. The last was determined in an Abbe refractometer. Basal, [Arg8]vasopressin-, and fluoride-stimulated activities of the 1,500 X g pellet layered on top of the gradient were 0.130, 0.260, and 0.560 nmole per 10 min per mg of protein, respectively.

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TABLE I

Partial puri$cation of renal medullary membranes enriched in NHH-sensitive adenylyl cyclase activity Adenylyl cyclase activities in the crude homogenate and the various fractions described under “Experimental Procedures” were

measured under standard assay conditions. When present, [Arg*]vasopressin was 10m7 M and NaF was 10 mM.

Fraction Volume

Adenylyl cyclase activity in the presence of [Arg*lvasopressin-

stimulated activity relative to basal

?d mglml

Crude homogenate 1560 2.25 0.034 0.065 0.111 1.91 S 150 1530 1.65 0.026 0.051 0.096 1.96 P 160 80 6.40 0.070 0.154 0.200 2.20

SlWl500 1520 1.50 0.007 0.010 0.029 1.42

P160-1500 200 4.00 0.146 0.268 0.445 1.83 32.5/37.0interface 58.5a 1.25 0.148 0.232 1.288 1.56 37.0/41.5 interface 88.4a 2.85 0.077 0.376 0.607 4.76 41.5 pellet 13.40 5.00 0.096 0.182 0.402 1.89

-

‘

.-

-

Increase in activity due to [Arg*lvasopressin

jpecific activity Total

tWtOlCS/lO n?noles/ min/mg 10 min

0.031 108.0 0.025 63.1 0.084 43.0 0.003 6.80 0.122 48.8 0.084 3.10 0.299 37.8 0.086 2.95

a Two consecutive discontinuous sucrose gradient centrifugations were carried out and the various interfaces and pellets from the 12 tubes pooled to give the listed volumes.

TIME imln ) PROTEIN (~‘9 per assay )

FIG. 2. Dependence of accumulation of CAMP on time of incu- bation and on concentration of renal medullary membranes. Assays were carried out under standard assay conditions. When present, [Arg*]vasopressin (AVP) was lo-’ M and NaF was 10 mM. Time courses were determined at 80 rg of membrane protein per assay. For rest of conditions, see “Experimental Procedures.”

[Arg*]vasopressin- and fluoride-stimulated) regardless of the time during which the membranes were kept frozen at -70” (longest time tested, 9 months).

The renal medullary membranes isolated by the method described under “Experimental Procedures” are by no means pure plasma membranes. Heavy contamination by mitochon- dria, endoplasmic reticulum, and other cell organelles was detected both by electron miscroscopy and by marker enzyme studies. In one representative study it was found that the specific activities (expressed per mg of protein) for glucose 6-phosphatase, 5’-nucleotidase, and cytochrome c reductase were, in the final membrane preparation, 0.94 pmole per hour, 0.15 pmole per hour, and 0.248 pmole per min, respectively, while those of the starting crude homogenate were 1.22 pmoles per hour, 0.38 pmole per hour and 0.045 /Imole per min, respectively. Thus the NHH-sensitive adenylyl cyclase-containing membranes fractionated closely with cytochrome c reductase, a mitochon- drial marker enzyme. Close association of mitochondria with plasma membranes in renal tubular cells has been pointed out by Fitzpatrick et al. (28).

As shown in Fig. 2, under standard assay conditions (1.0 mM

ATP and 5.0 mM total added MgC1.J the adenylyl cyclase reaction proceeded linearly with respect to time and to protein concentration, both in the absence and the presence of a maxi- mally stimulating concentration of [Arg*]vasopressin.

Dependence of NHH-stimulated Adenylyl Cyclase on Substrate and Magnesium Concentration

Initial experiments that tested the dependence of the enzyme system on magnesium ion showed that adenylyl cyclase activity was essentially undetectable at any concentration of added MgClz below that of added EDTA. Similar basal and NHH- stimulated activities were obtained at 2.0 mM MgC!lz in the presence of 1.4 mM EDTA as with 3.0 mM MgClz in the presence of 2.4 mM EDTA. We have therefore expressed magnesium ion concentrations in millimoles per liter (mM) in excess of the EDTA concentration present in the assay.

Studies with ATP

The effect of varying the concentration of ATP in the incuba- tion medium at different magnesium concentrations on the renal medullary membrane adenylyl cyclase, determined in the absence and presence of [Arg*]vasopressin, is shown in Fig. 3, A and B. Two main findings emerged from this study: (a) the basal, hormonally unstimulated activity of the system is stimu- lated by increasing concentration of Mg*+ more than the hor- monally stimulated activity, a fact that leads to decreasing hormone stimulation relative to basal with increasing concen- trabion of magnesium; and (b) at any given concentration of Mg2+, basal and hormonally stimulated activities respond differently to varying concentrations of ATP. This finding is further illustrated in Fig. 3C where the [Args]vasopressin-depend- ent stimulation is expressed relative to the respective basal activity. It can be seen that the enzyme system is maximally stimulated by [Arg*]vasopressin at concentrations of ATP ranging from 0.07 to 0.10 mM, and that at both higher and lower concentrations of ATP, the relative stimulation by [Arg*]vaso- pressin is less. Thus, while “standard” assay conditions of 1.0 mM ATP and 3.6 Mg2+ (in excess of 1.4 mM EDTA) give a relative stimulation of about 3- to 4-fold over basal, reduction of the concentrations of ATP and Mg2+ to 80 PM and 0.6 mM, respectively, results in an increase of the relative stimulation by [Aq$]vasopressin to 7- to g-fold over basal.

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.’ ‘1.6

1.0

ATP (mM) ATP (mM)

B. .

/-

26.6 .

FIG. 3. A and B, effect of ATP concentration on basal and [Argslvasopressin (AVP)-stimulated adenylyl cyclase activities determined at various Mg2+ concentrations (expressed as milli- moles per liter (mM) in excess of 1.4 mM EDTA). Activities were determined at pH 8.0. Membrane protein was 58 pg per assay. The specific activity of the [a-azP]ATP used as substrate was varied to give 10,032, 5,896, 2,414, 859, 320, 90, and 33 cpm per

AMP - PNP: 114,1M 1.0 ATP 90)M

on-

: . . . E$asa, 5 - AVP c ::

FIG. 4. pH dependence of basal and [Ar@]vasopressin (AVP)- stimulated activities (expressed in millimoles per liter (mM) in excess over 1.4 rnM EDTA) using ATP and AMP-PNP as sub- strate. Incubation conditions were those described under “Ex- perimental Procedures” except for the following changes: (a) the concentrations of 32P-labeled ATP (1000 cpm per pmole) and AMP-PNP (1800 cpm per pmole) were those shown on the figure; (b) the concentration of Buffer B was increased to 50 mM; and (c) the concentrations of Mg2+ were those shown on the figure next to the corresponding curves. The indicated pH values are actual values after 5 min of incubation. The nucleoside triphos- phate regenerating system was omitted from incubations with AMP-PNP. When present, [Argslvasopressin was lo-’ M; mem- brane protein was 55 rg per assay in the experiment with ATP and 47 pg per assay in the experiment with AMP-PNP.

Effect of pH at Different Concentrations of MS*+---At low con- centrations of Mg2+ (0.6 mm), the pH optimum of both basal and [Ar$]vasopressin-stimulated adenylyl cyclase activities is broad, extending from pH 8.0 to 9.5. Increasing concentrations of Mg”+ result in a narrower pH curve with a maximum between pH 7.8 and 8.0 and the appearance of an inhibitory effect of Mg*+ at pH values exceeding 9.6 (see Fig. 4, right).

Studies with AMP-PNP

This nucleotide was first shown to be a substrate for adenylyl cyclase activity in liver plasma membranes by Rodbell et al. in 1971 (7) and subsequently also in human platelet membranes (29) and more recently in fat cell membranes (30). It was therefore of interest to see whether also the renal medullary

6.6

3.6 26.6

6.6

3.6

-

ZI 12.0 c .1 t5 10.0

Q8 D m 6.0 LO 0- 6.0

.: .z ZS 4.0

Ii lf 3

2.0 1.0

0 1 I , (Basal= 1.0)

0.01 0.1 1.0

ATP(mM) pmole at 0.0058,0.0107,0.077,0.216,0.72 and 2.33 mM, respectively. - - -, basal activities; -, activities obtained in the presence of lo-’ M [Ar@]vasopressin; numbers represent Mg2+ concentrations. For rest of conditions, see “Experimental Procedures.” C, [Ar$]vasopressin-stimulated activities shown in A and B are expressed relative to their respective basal activities.

Ma*+: 3.6 mM

IP-PNP(mM) ATP (mM1

FIG. 5. Comparison of AMP-PNP-driven and ATP-driven adenylyl cyclase reactions assayed under identical conditions. Activities with AMP-PNP were assayed in the absence (-RS) and the presence (+RS) of the standard nucleoside triphosphate regenerating system consisting of 0.2 mg per ml of creatine kinase and 20 mM creatine phosphate. Activities with ATP were assayed only in the presence of the regenerating system. All activities were determined at pH 8.0. The specific activity of the [a-82P]- ATP was varied with the concentration used to give 2,600,202, 164,154, and 52 cpm per pmole at 0.07,0.43,0.85,1.49 and 2.56 mM, respectively. The specific activity [c?~P]AMP-PNP was varied with the concentration used to give 2,103,1,195,808, 448,367,272, 183, and 84 cpm per pmole at 0.06, 0.117,0.233, 0.427, 0.626, 0.826, 1.266, and 2.50 mM, respectively. Membrane protein was 33 rg per assay. [Arglvasopressin (AVP), when present, was lo-’ M. Mg2+ concentration is expressed in excess over 1.4 mM EDTA. For rest of conditions, see “Experimental Procedures.” Basal and [Ar@]vasopressin-stimulated activities with AMP-PNP in the presence of the nucleoside triphosphate regenerating system (left) were replotted on the right for comparison, using the same activity scale used to represent activities obtained with ATP.

membrane adenylyl cyclase system reacted with this synthetic analogue of ATP and if so, how it compared to ATP, a compari- son which has not yet been made in other systems. It was found that the synthetic nucleotide is also a substrate for this adenylyl cyclase. A study of the dependence of the renal adenylyl cyclase system on AMP-PNP concentration, carried out both in the absence and presence of a nucleoside triphosphate regenerating system and comparison to the dependence of the system on ATP concentration is illustrated in Fig. 5. In this and other studies it was established that although AMP-PNP is capable of supporting the adenylyl cyclase reaction and allows for hormonal stimulation, the absolute adenylyl cyclase activities

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zI 0.25 C .z '5 a

0.20

i% 0 0.15 3 a " 0.10

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! j j 0.05

%

0

A. / 5

' 1.6

AMP-PNP(mM)

0.0

0.6

0.4

0.2

9

B

0.01 011

AMP-PNP (mM)

FIG. 6. A and B, effect of AMP-PNP concentration on basal and [Args]vasopressin (AVP)-stimulated adenylyl cyclase activ- ities determined at various Mgf+ concentrations (expressed as millimoles per liter (mM) in excess over 1.4 mM EDTA). Activ- ities were determined at pH 9.0. The specific activities of the [w~~P]AMP-PNP used as substrate was varied to give 3,190, 3,190, 3,190, 3,703, 2,661, 1,725, 1,187, 623, 347, and 183 cpm per

that can be obtained with the synthetic substrate are significantly less than those that are obtained under similar conditions using ATP, regardless of whether the AMP-PNP reaction is carried out in the absence or t,he presence of a nucleoside triphosphate regenerating system. In the particular experiment shown in Fig. 5 basal and [Args]vasopressin-stimulated activities driven by AMP-PNP were about 50 and 20%, respectively, of those driven by ATP, varying somewhat with the concentration of substrate used.

Effect of pH-At 0.6 mM Mg +- the AMP-PNP-driven basal adenylyl cyclase activity has a pH optimum of 9.6. This pH optimum is shifted toward lower values with increasing concen- trations of Mg2f so that at 18.6 mM the pH optimum is 8.3. Similarly, in the presence of [Arg8]vasopressin, the pH optimum shifts from 9.0 at 0.6 mM Mg2f to 8.3 at 18.6 mM Mg2+. The activities obtained with AMP-PNP were less than those obtained with ATP under all conditions tested. These findings are illustrated in Fig. 4.

Effect of AMP-PNP and Mg2+ Concentration on Stimulation of Renal Adenylyl Cyclase by AVP-Using AMP-PNP (pH 9.0, absence of regenerating system) the dependence of the system on Mg2+ followed a pattern similar to that seen with ATP, i.e. the basal activity is stimulated to a greater extent by increasing concentrations of the divalent action than the [Arg*]vasopressin- stimulated activity. The dependence of the system on AMP-PNP concentration, however, differed from that seen with ATP in that (a) hormonal stimulation required, to be detectable, the presence of at least 0.05 mM AMP-PNP, and (b) no inhibition of hormonal stimulation (relative to basal) was observed with concentrations of AMP-PNP up to 1.0 mM, regardless of the Mg2+ concentration used. This is illustrated in Fig. 6. In other experiments no inhibition by excess AMP-PNP was seen even at concentrations as high as 2.5 mM, which is about 20-fold higher than the concentration of ATP at which a clear diminu- tion of hormone action can be detected.

Stimulation of Renal Medullary Membranes by Other Neurohypophyseal Hormones and

Prostaglandin E,

Under conditions that were found optimal for studying the response to [Arg*]vasopressin, renal medullary adenylyl cyclase activity is also stimulated by two other natural neurohypophy-

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AMP-PNP(mM)

pmole at 0.0042, 0.0097, 0.0197, 0.046, 0.057, 0.086, 0.129, 0.232, 0.417, and 0.81 mM, respectively. - - -, basal activities; -, activities obtained in the presence of 10-r M [Arg*]vasopressin; numbers represent Mg2+ concentrations. 33 pg per assay.

Membrane protein was For rest of conditions, see “Experimental Proce-

dures.” C, [Ar&]vasopressin-stimulated activities shown in A and B expressed relative to their respective basal activities.

ATP: 78uM

0.6mM

Q 0 I 1 I III I I III I I III I I III I I III I I III I 1111

0 lo-” 10-10 10-g 10-e 10-7 10-6 10-5 10-4

NHH (M)

FIG. 7. Effect of varying concentrations of [Ar&]vasopressin (AVP), [Lys8]vasopressin (LVP), and oxvtocin (OT) on renal medullary adenylyl cyclase activity determined under conditions of ATP and M@ concentration oatimal for hormonal resaonse. Vertical lines indicate the concentrations of NHH at which 507, of maximal activation (apparent K, of activation) was obtained. Specific activity of the [w~~P]ATP used was 512 cpm per pmole; membrane protein concentration was 34 pg per assay. For rest of conditions see “Experimental Procedures.”

seal hormones, [Lys*]vasopressin and oxytocin. Both hormones stimulate adenylyl cyclase activity to the same extent as [Arg*]- vasopressin. As illustrated in Fig. 7 the beef renal medullary adenylyl cyclase system is equally sensitive to [Args]vasopressin and [Lys8]vasopressin and about 100 times less to oxytocin. The apparent K, values of activation (apparent K,) for the three NHHs varied somewhat with the batch of renal medullary membranes tested. Thus the apparent K, for [Arg8]vasopressin determined in seven membrane preparations varied between 0.8 and 3 x 1O-g M with a mean of 1.25 x 10eg M, and that for oxytocin, determined in four membrane preparations, varied between 0.5 and 2 X 10e7 M with a mean of 1 X lo-’ M. In the particular experiment shown in Fig. 7, concentrations of [ArgS]- vasopressin, [Lys8]vasopressin, and oxytocin giving half-maximal stimulation of adenylyl cyclase activity (apparent K, values) were 1.1, 1.1, and 50 X 1O-g M respectively.

As shown in Table II, renal medullary membranes also contain an adenylyl cyclase system that responds to PGEi. Activation

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TABLE II

Effect of [Arga]vasopressin and PGE, on renal medullary adenylyl cyclase activity

Renal medullary adenylyl cyclase activity was determined under standard assay conditions. When present, [Arg8]vaso- pressin was IO-’ M and PGEr was 3 X 10m6 M. Membrane protein was 36 rg per assay. For rest of conditions see “Experimental Procedures.”

Additions Adenylyl cyclase activity’”

Change in adenylyl cyclase activity due to addition of

Lh*l- vasopressin PGEl

None lArg*]vasopressin PGE, 1 iEi ~ 2g4 1 146 [Argslvasopressin +

PGE,

a Values are mean f standard deviation of quadruplicate de- terminations and are expressed in picomoles of CAMP formed per 10 min per mg of membrane protein.

TABLE III

E$ect of GTP on renal medullary adenylyl cyclase activity and its response to [Argalvasopressin

Adenylyl cyclase activity was determined at 0.087 mM ATP (736 cpm per pmole) and 0.6 mM Mg2+ (excess over 1.4 mM EDTA). When present, [Arg8]vasopressin was 10e7 M and GTP was 10m5 M.

Membrane protein was 62 pg per assay. For the rest of the condi- tions see “Experimental Procedures.”

Additions Adenylyl cyclase activity0 AVP-stimulated

activity rela.tive to -[Arg*]vasopressin +[Arg*jvasopressin

basal”

None 67 f 3 445 + 6 6.65 GTP 80 xt 2 275 f 4 3.44

a Values are mean f standard deviation of quadruplicate de- terminations and are expressed in picomoles of CAMP formed per 10 min per mg of membrane protein.

b Ratio of activation is obtained by dividing adenylyl cyclase activity obtained in the presence of 1O-7 M [Arg8]vasopressin by that obtained in the absence of [Args]vasopressin.

by maximally stimulating concentrations of [Arg8]vasopressin (lo-’ M) and of PGEi (3 X lo+ M) were additive.

E$ect of GTP-This nucleotide has been shown in several adenylyl cyclase systems to enhance, promote or stimulate the action of hormones (7, 29-33). As shown in Table III, addition of 10m5 M, GTP to the incubation medium resulted in inhibition, rather than stimulation of [Arg8]vasopressin response.

DISCUSSION

The methodology described in this report for the preparation of renal medullary membranes has several advantages. (4 It gives a high yield of membranes (approximately 250 mg of membrane protein) and is therefore adequate for future prepara- tive scale work dealing with isolation of components. (5) It separates the NHH-sensitive adenylyl cyclase system from other adenylyl cyclases not, sensitive to these hormones and which account for up to two-thirds of the total renal medullary adenylyl cyclase activity (see Table I). (c) It yields a NHH-sensitive system that is both highly responsive (up to lo-fold stimulation under adequate assay conditions) and highly sensitive (ap-

parent K, of activation for [Arg8]vasopressin, low9 M). (d) It

yields a NHH-sensitive system that has preserved some of its regulatory properties as seen by its complex modulation by ATP and Mg2+, and by its response to GTP. It should be mentioned that to successfully obtain the membrane fraction enriched in NHH-sensitive adenylyl cyclase it was necessary to (a) include 1 mM EDTA in sucrose gradients used for separation, and (5) have gone through a hypotonic dilution step before preparing the low speed particulate fraction (P~M)-~,sw) used as source of adenylyl cyclase in the sucrose gradient separation. Selective enrichment with NHH-sensitive adenylyl cyclase could not be obtained when the homogenate was prepared in either isotonic (0.25 M) or hypertonic (0.8 Ml sucrose, be it in the absence or presence of 10 mM MgC12, 1.0 mM EDTA, 10 mM Tris-HCl, pH 7.5, or combinations thereof. While this and subsequent studies on the beef renal adenylyl cyclase system were well in progress, Finn et al. (34) working with beef adrenals and using a zonal centrifugation technique published a method for separating mitochondrial membranes and fragments from adrenocortico- tropic hormone-sensitive adenylyl cyclase-containing membranes. We have not yet tested whether the method of Finn et al. is also applicable to our renal medullary membrane preparations; however, in view of the presence of large amounts of mito- chondrial fragments in our renal medullary membrane prepara- tion, ultimate inclusion of a rate zonal centrifugation step into the purification scheme may well be advantageous. We are currently investigating this possibility of improving the relative purity of NHH-sensitive adenylyl cyclase-containing membranes.

One striking property of our preparation of renal medullary membranes is that its NHH-sensitive adenylyl cyclase system is profoundly influenced by -4TP and Mg2+, to the extent that, to establish optimal condition for the assay of NHH-dependent stimulation, it became essential to carry out an extensive study in which both substances were varied simultaneously. From this study it became apparent that hormonal stimulation is best seen at low Mg2+ concentrations and depends on an optimal concentration of ATP. Recently three other reports have appeared dealing with the partial purification of renal medullary membranes. Campbell et al. (35) prepared membranes from pork renal medulla using a differential centrifugation method described by Fitzpatrick (28) for the preparation of ATPase- enriched membrane fractions, and obtained a NHH-sensitive adenylyl cyclase system that is 6-fold purified with respect to the starting homogenate and which responds to [LysJ]vasopressin with a 3- to 3.5.fold increase in activity when determined at 1.2 mM ATP and 6.6 mM MgC&; half-maximal response to [Lys8]- vasopressin was found at 2.2 x lo-* M. Neer (36) prepared membranes from rat renal medulla and obtained a NHH- sensitive system that is 5-fold purified with respect to the starting homogenate and responds with a 6-fold increase in activity when determined at about 1.0 mM ATP and 9.0 mM MgC&; half- maximal stimulation by [Lys8]vasopressin varied for different membrane preparation between 4 X 1O-g M and 15 X 1O-g M.

Finally, Bockaert et al. (37) have also partially purified mem- branes from pork renal medulla using the technique described by Emmelot et al. (38) for liver plasma membranes and obtained a NHH-sensitive system that is about i-fold purified with respect to the starting homogenate and responds to [Lys8]vasopressin with a 5-fold stimulation when determined at 0.25 mM ATP and 10 mM MgC12; half-maximal stimulation by [Lys8]vasopressin was found at 5 X 1O-g M. In none of these studies was a regu- latory effect of either Mgaf or ATP reported that resembled our findings. On the contrary, the rat renal medullary system

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appeared to follow Michaelis-Menten kinetics for all three activities tested (basal, [Lys*]vasopressin- and fluouride-stimu- lated), which ours did not. The reason for this difference is not clear, but may probably be found in the method of prepara- tion of membrane fractions which may alter the regulatory properties of the adenylyl cyclase system(s) embedded in them. It is possible that in these studies some of the ATP-dependent effects seen in our preparation were masked by the presence of NHH-insensitive adenylyl cyclases that significantly contribute to the experimentally determined basal activity. Neither in the study by Campbell et al. (351, nor in those of Neer (36) or Bockaert et al. (37), has the presence of other hormone-sensitive adenylyl cyclase systems in the final membrane preparation been tested. In this sense it should be pointed out that even though our adenylyl cyclase preparation is selectively enriched with respect to the NHH-sensitive system, it is not free from that complication either; there is at least one other adenylyl cyclase system present that responds to PGEi and whose basal activity must be contributing to some extent to the total basal activity determined in the adenylyl cyclase assay. The recent report by Storm and Dolginow (39), demonstrating a glucagon- stimulated inactivation of the glucagon-sensitive adenylyl cyclase activity in liver plasma membranes by iodoacetamide may have provided a tool to explore quantitatively the question of relative contribution of several adenylyl cyclase systems to “basal activity.”

Relatively low adenylyl cyclase activities were obtained with AMP-PNP as substrate. Two possible explanations come to mind. One relates to the fact that the products of the reaction catalyzed by adenylyl cyclase are CAMP and PNPi when AMP-PNP is used as substrate as opposed to CAMP plus PPi that are the natural products obtained with ATP. It is not known which is the rate-limiting step of the reaction catalyzed by adenylyl cyclase, but if the exit of PNPi is sufficiently slow

to either become the rate-limiting step or is slower than an already rate-limiting exit of PPi, then a slower product removal from the cat.alytic site would be one explanation for the lower activities observed with AMP-PNP. Another explanation is based on the chemical properties of AMP-PNP itself. Yount et al. (18) have demonstrated at least two important charac- teristics of AMP-PNP to be different from those of ATP. (a) Under conditions of maximal ionization, i.e. above pH 8.5 where both ATP and AMP-PNP exist mainly in their -4 net charge form, AMP-PNP binds Mg*+ only one-half as strongly as ATP. (b) The pK of the terminal phosphate of AMP-PNP is 7.7 as opposed to 7.1 in ATP. Both differences tend to de- crease the fraction of AMP-PNP in the bound form, substrate for adenylyl cyclase, and to increase proportionately the free form of the nucleotide, inhibitory to the reaction (1, 40), and therefore conspire against optimal catalytic activities. The finding of a higher pH optimum for basal adenylyl cyclase activity using AMP-PNP as substrate is consistent with this hypothesis. These explanations are, of course, speculative and a definitive answer will have to come from studies with a purified system exploring the mechanism of the catlaytic process converting ATP (or AMP-PNP) to CAMP plus PPi (or PNPi).

Adenylyl cyclases are stimulated by an active hormone receptor. Studies from this and other laboratories have estab- lished that this stimulation is not only dependent on the forma- tion of a hormone-receptor complex, but also on the presence or absence of certain nucleotides. In this study (Fig. 3) we found that ATP exerts two effects on the NHH receptor-dependent activation of beef renal medullary adenylyl cyclase. One effect,

7855

seen when concentrations of ATP are increased from 10e6 M to 10e4 M, results in enhancement of hormonal stimulation; the other effect, seen when concentrations of ATP are increased from lop4 M to 1OV M, results in inhibition of hormonal stimu- lation.. This second effect is not seen with AMP-PNP as substrate (see Fig. 6). These findings suggested that activation of adenylyl cyclases by hormone receptors may very well be under control by nucleotides in a positive manner, as seen in earlier studies with glucagon-sensitive adenylyl cyclases from liver and pancreatic fl cells, as well as in a negative manner as suggested by the inhibitory effect of high concentrations of ATP on NHH stimulation of the renal medullary adenylyl cyclase. The finding that GTP at 1OV mimicked the effect of millimolar concentrations of ATP supports this hypothesis. A detailed study on the characteristics of the GTP effect as well as that of adenosine, found to mimic the stimulatory effect of low concen- trations of ATP, is reported in the accompanying paper.

In conclusion, this report describes the method of preparation of a beef renal medullary membrane fraction in relatively large quantities and the basic properties of a NHH-sensitive adenylyl cyclase system present in these membranes. These membranes have subsequently been used to study the regulation of the receptor dependent activation of adenylyl cyclase by nucleosides and nucleotides, to determine rate constants of the hormone- receptor interaction as seen through activation of adenylyl cyclase, and to define several conformational states of the cata- lytic unit of the system as induced by the free receptor, the NHH-occupied receptor and GTP. These studies are the subject of some of the accompanying reports (41-43), all dealing with the mechanism(s) by which hormone receptors affect adenylyl cyclase systems.

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Lutz Birnbaumer and Po-Chang YangSENSITIVE TO NEUROHYPOPHYSEAL HORMONES

CYCLASE SYSTEM IN BEEF RENAL MEDULLARY MEMBRANESAND DESCRIPTION OF GENERAL PROPERTIES OF AN ADENYLYL

Studies on Receptor-mediated Activation of Adenylyl Cyclases: I. PREPARATION

1974, 249:7848-7856.J. Biol. Chem. 

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