168, 455-462 (197.5)

of Solubilized Liver Membrane by Guanyl Nucleotides’ NORBERT Memorial








Cancer Center, New York, New York 10021

Received December

23. 1974

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 M Lubrol-PX and centrifuged at 165,OOOg for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mM ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/l0 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mM nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.

The role of adenylate cyclase in mediating hormone action at the plasma membrane is well recognized (1). It is becoming increasingly apparent that guanyl nucleotides can modify adenylate cyclase activity, hormone binding to membranes, and adenylate cyclase activation by hormones. In liver plasma membranes adenylate cyclase sensitivity to glucagon (2, 3), and to epinephrine (3, 4), was enhanced by GTP and other nucleotides. Similar effects of nucleotides on adenylate cyclase have been observed with islet cell tumors (5) and 1 From Memorial Sloan-Kettering Cancer Center. This investigation was supported in part by Research Grant CA-08748 of the National Institutes of Health, Grant GB-19797 of the National Science Foundation, and Grant BC-119 of the American Cancer Society. A portion of this study has been presented in abstract form, Second International Conference on Cyclic AMP, 1974, Vancouver, British Columbia. 2 To whom requests for reprints should be made. 455 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

islets (6, 7), toad bladder epithelial cells (a), platelets (9), thyroid gland membranes (10, ll), turkey erythrocytes (12), adipocytes (13, 14), and adipocyte membranes (13, 14). It has been proposed (2, 3, 6, 9, 15, 16) that nucleotides or products of nucleotide metabolism (17, 18) bind to sites on the membrane and have regulatory effects on catalytic subunits of the enzyme. Adenylate cyclase has been solubilized from several tissues by Lubrol-PX, a nonionic detergent (19, 20). We have reported (21) that treatment of liver plasma membranes with Lubrol-PX, yielded a solubilized adenylate cyclase preparation that retained sensitivity to GTP, ITP, and GMP-PCP.3 Pilkis and Johnson (22), however, were not able to demonstrate a stimulation of Lubrol-solubilized liver mem3 The following abbreviations 5’-guanylylmethylenediphosphonate; guanylylimidodiphosphate.

are used: GMP-PCP, GMP-PNP, 5’-



brane adenylate cyclase with GTP. The present report extends our earlier observations. METHODS Liver membranes of hypophysectomized rats, obtained from Charles River Breeding Farms, were isolated by a modification (17) of the Neville (23) procedures. Membranes were obtained which in our laboratory and elsewhere (24) are responsive to glucagon. Membranes have been characterized by marker enzyme activity and electron microscopy in previous communications from this laboratory (17, 18). After preparation, the membranes were stored overnight in 30% sucrose at 4°C and used on the following day. Adenylate cyclase activity was assayed by the method of Krishna et al. (25) in a total volume of 0.1 ml in 50 mM Tris:HCl buffer, pH 7.4 with 2 mM EDTA and 10 mM MgCl, with 2-5 PCi [(Y-~*P]ATP, obtained from International Chemical and Nuclear Corp., and an ATP regenerating system (24). After incubation 0.5 mg cyclic AMP was added to each tube and the reactions were terminated by boiling for 3.5 min. Cyclic [a-32P]AMP was purified by Dowex-50 H+ chromatography and ZnSO,-Ba(OH), precipitation. Recovery of cyclic AMP was determined spectra: photometrically at 260 nm after addition of cyclic AMP (20). Proteins were determined by the method of Lowry et al. (26). Adenylate cyclase activity from liver plasma membranes was solubilized as follows. A sample of the membrane containing from 3 to 6 mg protein was washed in 10 ml of 1 mM NaHCO, and 0.5 mM CaCl, and centrifuged at 35,000g for 20 min. The pellet was resuspended in 0.2 M Tris-HCl, pH 7.4, containing 0.1 M Lubrol-PX, 0.25 M sucrose, 1 mM EDTA and this was centrifuged at 165,OOOg for 1 h. Two-hour centrifugation did not alter significantly the yield or specific activity of the solubilized enzyme. A clear supernate was obtained which contained solubilized adenylate cyclase. Activity of the solubilized enzyme was linear between 4.0 and 42.5 pg protein. The product of the enzyme reaction cochromatographed with authentic cyclic AMP as determined by paper (27) and thin layer (28) chromatography. For the analysis of nucleotide hydrolysis, thin layer chromatography of reaction mixtures was performed by the procedure of Randerath and Randerath (28) on PEI-cellulose plates (Brinkmann) except that after spotting the plates were run in water and dried before the final development in LiCl. This procedure increased the separation of nucleotide metabolites. After drying, the lanes corresponding to different samples were cut into l-cm segments and radioactivity thereon determined by liquid scintillation counting. All nucleotides except GMP-PCP were obtained from P-L Labs. GMP-PCP was obtained from Sigma and [3H]GMP-PCP was generously provided by Drs.

AND TIERNEY Herbert Weissbach and David Miller. “C-Labeled nucleotides were purchased from Schwartz. RESULTS

Treatment of liver plasma membranes with Lubrol-PX yielded a soluble adenylate cyclase preparation that was not sedimentable by centrifugation at 165,000g for 2 h. Activity of the soluble enzyme was linear from 4 to 42.5 pg protein when assays were conducted for 10 min. Approximately 50% of the membrane protein was solubilized with the detergent, The specific activity of the solubilized enzyme varied as a function of substrate concentration when direct comparisons are made to the membrane enzyme (Table I). With equal Mg2+ concentrations (10 mM) the solubilized enzyme possessed different substrate requirements for optimal activity than the membrane enzyme from which it was derived (Table I). Membrane enzyme substrate optimum was similar to that described by Pohl et al. (24). Lower substrate concentrations were required for optimal activity of the soluble enzyme. The soluble form exhibited maximal activity at 0.32 mM ATP, a substrate optimum one order of magnitude lower than that required for the membrane enzyme. At its substrate optimum the soluble enzyme exhibited 11% of the specific activity of the membranes. Nucleotide activation of the solubilized adenylate cyclase was examined at the TABLE



Membranes 165,OOOg supernate






69.9 8.2

519.0 5.0

372.2 39.5

24.6 12.5

1.03 2.0

a The membrane preparation was solubilized in 0.1 M Lubrol-PX in 0.2 M Tris-HCl, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA. Adenylate cyclase was assayed in 0.1 ml 50 mM Tris-HCl, pH 7.4, 2 mM EDTA and 10 mM MgCl,, with an ATP regenerating system of 1 mg creatine phosphate and 50 units creatine phosphokinase, with 2 PCi [a-32P]ATP per sample.



substrate optimum of the soluble enzyme. As seen in Table II, activation with 0.1 mM nucleotides occurred in the following order of effectiveness GMP-PCP > ITP >GTP. For the membranes, the order of activation was GMP-PCP > GTP > ITP. The solubilized enzyme exhibited sensitivity to GTP, GMP-PCP, and ITP whether assays were conducted at 0.32 mM or 3.2 PM. The lower, suboptimal concentration was examined since Goldfine et al. (5) reported that GTP maintained activity of p-cell adenylate cyclase independent of ATP concentrations from 1 mM ATP to 1.3 PM ATP. While the absolute activity of the enzyme decreased loo-fold at the lower substrate concentration the relative activation by added nucleotides was in the order GMP-PCP > ITP > GTP (Table III), which was similar to that observed with the soluble enzyme assayed at the optimal substrate concentration (Table II). The addition of 0.5 InM Ca2+ to the solubilized enzyme (Table III) increased the activity of basal and nucleotide activated enzyme by 29-40%. The order of activation by the nucleotides with added Ca2+ remained the same as observed without Ca2+, GMP-PCP > ITP > GTP. GMP-PCP







tent nucleotides of several examined, in increasing adenylate cyclase activity (Table IV). At 0.1 mM added nucleotide, fivefold and fourfold increases were observed (Table IV) for GMP-PCP and GDP, respectively. Other guanine derivatives were less effective. At 1 PM the guanyl nucleotides were without effect on the solubilized enzyme. To examine the possibility that increased activity of the adenylate cyclase in the presence of guanyl nucleotides was due to a sparing effect of the added nucleotides on hydrolysis of substrate ATP by ATPases, thin layer chromatographic separations of the reaction mixtures, containing cu-32P-labeled substrate ATP and unlabeled nucleotides, were performed and were analyzed by liquid scintillation counting. Our results (Table IV) indicate that 85-93% of the substrate ATP was recovered after assay whether or not adenylate cyclase activation was observed. Moreover,







pm01CAMP/ mg/lO min 165,OOOgsupernate 165,000g supernate + GMP-PCP 165,000g supernate + GTP 165,000g supernate + ITP Membranes Membranes + GMP-PCP Membranes + GTP Membranes + ITP

111.03 237.85 126.24 144.22 350.18 503.98 455.09 378.62

* 8.30 zt 14.93 * 12.13 * 8.13 * 11.05 i 2.87 * 19.90 +z 19.37

a The membranes were prepared and solubilized as indicated in text and in legend to Table I except that the membranes were washed free of Ca2+. Samples of 27 and 34 pg protein were used for the solubilized and the particulate enzymes, respectively. They were assayed with 0.32 mM ATP as described with 3.0 PCi [w~“P]ATP. Nucleotides were added just prior to assay at 0.1 mM. TABLE



pmol cAMP/mg/lO

165,000g supernate 165,000g supernate GMP-PCP 165,OOOgsupernate ITP 165,OOOgsupernate GTP



No Ca2+

+0.5 mM Ca2+


1.41 i 0.06 3.99 + 0.18

1.98 + 0.05 5.10 * 0.10


2.92 * 0.07

3.80 + 0.08


2.05 * 0.08

2.73 * 0.09

n Adenylate cyclase was solubilized and assayed with 3.2 PM ATP as described in text and legend to Table I with the Ca’+ washed out as described in legend to Table II. The protein concentration was 27 pg and the nucleotides, at a concentration of 0.1 mM, were added just prior to assay with 3.0 &i [a=P]ATP.

while final substrate ATP concentrations among the adenylate cyclase samples assayed with and without 0.1 mM guanyl nucleotides was 89.9 * 3.0%, adenylate cyclase activity was increased by as much as fivefold. With the solubilized enzyme obtained from liver membranes GMP-PCP from 1 PM to 1 mM was the most effective nucleotide examined in increasing adenylate cy-



clase activity (Fig. 1). ITP was also effective in this regard but to a lesser extent than GMP-PCP. GTP was the least effective nucleotide. The effects of GTP and ITP were most pronounced at 0.1 mM and 1




The liver plasma membranes and the solubilized material possess nucleotide phosphohydrolases (Table V). With memTABLE



pm01 cAMP/mg/lO No nucleotide GMP-PCP GTP GDP GMP Guanosine

35.38 192.75 46.19 153.51 80.94 24.70 -

Mean + SD

% ATP min

+ 17.5 * 37.3 + 7.1 l 30.3 * 15.8 + 15.8

89.4 84.5 92.8

91.0 89.3 92.4 89.9 i 3.0

u Adenylate cyclase was solubilized and assayed with 0.32 mM ATP as described in the text and legend to Table I. The nucleotide concentration was 0.1 mM, with enzyme concentration of 33 fig protein per sample. The amount of ATP remaining after assay was determined by thin-layer chromatography on PEI-cellulose by step-wise development with lithium chloride (28).



FIG. 1. Effects of nucleotides on adenylate cyclase activity. Solubilized enzyme was prepared as described in legend to Table I. Assays were conducted in 0.32 FM ATP with 2 HCi [,-32P]ATP per sample. Nucleotide concentrations were as indicated.

GTP Membranes 165,000~ Supernate


ATP regen:ratinf jysterr

GDP Membranes 165,OOOg Supernate






1.18 34.4 21.8 72.3

13.5 7.7 8.9 5.5

21.3 8.0 16.1 2.8

56.3 42.6 44.7 13.3

+ +

1.9 41.4 26.3 81.8

18.5 4.7 16.3 3.9

24.2 11.4 10.0 2.5

54.9 40.6 42.7 10.3

+ +

50.2 62.3 70 76

0 0 0 0

6.7 0 0 0

25.0 22.1 21.8 11.3

0.2 16.1 0.4 63.1

4.4 2.2 15.4 1.5

11.4 13.5 54.4 5.1

83.2 67.2 28.9 23.2


GMP-PCP Membranes 165,000~ Supernate

‘ercent nucleotide



ITP Membranes 165,OOO.a Supernate



+ +

a Membranes and solubilized adenylate cyclase, at protein concentrations of 45 and 29 rg, respectively, were prepared and assayed as described in the text and in legend to Table I. They were assayed with and without the ATP regenerating system and with labelled nucleotides at a concentration of 0.1 mM. The products of hydrolysis were identified by thin-layer chromatography on PEI-cellulose plates using the lithium chloride stepwise solvent system of Randerath and Randerath (28). R-PPP. R-PP. R-P, and R designate the nucleoside triphosphate, -diphosphate, -monophosphate, and riboside derivatives.

branes, GTP and ITP were hydrolyzed by 56% to their respective ribosides. In the presence of the ATP regenerating system the degree of breakdown was reduced by 15%. GMP-PCP was more resistant to hydrolysis by membranes as only 25% of the original material was converted to guanosine. GDP was the most labile nucleotide examined. Without the ATP regenerating system 83% of GDP was hydrolyzed. In all instances the ATP regenerat-



GTP concentrations as much as 90% of the original ATP was recovered (cf. Table IV). After the foregoing experiments were completed and while this manuscript was in preparation Londos et al. (29) reported that GMP-PNP was a potent activator of adenylate cyclase in membranes of eukaryotic cells. As seen in Table VII GMP-PNP also activated the soluble form of the enzyme. The degree of activation was similar to that observed with GMP-PCP. Both GTP analogs were four times as potent as the naturally occurring nucleotide.

ing system aided in restoring in part, the levels of added guanyl nucleotides. Solubilization of the liver membranes with Lubrol-PX did not qualitatively alter phosphohydrolase activity. GTP and ITP were hydrolyzed to their ribosides by over 40%. The regenerating system protected the nucleoside triphosphates from the hydrolysis by 50%. GMP-PCP was hydrolyzed by the solubilized enzyme as was observed by the membranes. GDP hydrolysis to guanosine by the soluble enzyme was reduced by the ATP regenerating system; most of the products of GDP hydrolysis were identified as GMP. Without GTP, 0.32 mM [(y-32P]ATP was hydrolyzed to 36.0% ADP and 0.3% AMP by membranes, and by 31.0% to AMP by the solubilized membrane supernate, when incubated for 10 min under adenylate cyclase assay conditions. The membranes hydrolyzed [(Y-~*P]ATP to ADP, whereas the solubilized membrane fraction carried the hydrolysis to AMP. At low concentrations, little ATP remained after incubation either with membranes or solubilized enzyme (Table VI). Increasing additions of GTP up to 1 mM decreased the amount of ATP hydrolyzed both by membranes and the 165,000g supernate. Less GTP was required to maintain ATP concentrations as ATP concentrations were increased. At higher ATP and


The present findings extend our previous observations that a solubilized nucleotide sensitive adenylate cyclase can be preTABLE





GMP-PNP Nucleotides

pmol cAMP/mg/lO


124.1 248.9 239.6 156.8

a The with 0.32 to Table mM with sample.




i i zt zt


16.0 11.8 34.7 15.7

membranes were solubilized and assayed mM ATP as described in the text and legend I. The concentration of nucleotides was 0.1 enzyme concentrations of 30 pg protein per





GTP None


10 /.tM

0.1 mM

1 mh4

Percent ATP remaining 3.2 /.LM 32 PM 0.32 mM 3.2 mM

Membranes 165,OOOgsupernate Membranes 165,OOOgsupernate Membranes 165,OOOgsupernate Membranes 165,OOOgsupernate

6.8 22.2 8.0 32.6 60.9 64.7 87.6 91.6

4.4 28.0 8.2 37.7 57.6 12.7 85.5 92.4

5.6 35.4 7.8 47.7 62.0 80.9 83.3 92.6

7.8 48.5 16.7 65.9 69.3 83.9 90.4 89.4

60.8 83.4 74.4 84.4 81.6 88.7 90.7 93.2

” The membranes and solubilized adenylate cyclase were prepared as described in the text and in the legend to Table I with 3 ,.Xi [w~~P]ATP. The protein concentration was 56 pg for the membrane and 28 pg for the solubilized enzyme. The percent of ATP remaining was calculated after thin-layer chromatographic separation of the incubated material by the method described in the legend to Table V.



pared from liver plasma membranes by Lubrol-PX treatment. This report describes the preparation and some properties of a detergent-solubilized adenylate cyclase from rat liver membranes which retained sensitivity to activation by nucleotides. The substrate optimum of the soluble enzyme was one order of magnitude lower than had been observed for membrane adenylate cyclase by Pohl et al. (24) and as prepared here. Specificity of nucleotide sensitivity of the solubilized enzyme was similar to the membrane bound form. Enzyme activation of the solubilized adenylate cyclase was obtained with nucleotide concentrations which have been employed by others (2-16, 29) to demonstrate activation of the enzyme in membranes isolated from many different sources. Our earlier findings (21) and those reported here, differ with those of Pilkis and Johnson (22) who were unable to demonstrate sensitivity of the Lubrol-dispersed liver membrane enzyme to GTP. The differences may reside in the fact that GTP used at low concentrations (concentration not reported) by Pilkis and Johnson (22) was, in view of the present findings, probably rapidly hydrolyzed and present in insufficient amounts to be effective. The soluble adenylate cyclase retained sensitivity to fluoride (30), findings which are in agreement with the report of Pilkis and Johnson (1974) who also observed that fluoride stabilized the enzyme during incubation at 37°C for 40 min. The rat kidney enzyme solubilized with Lubrol-WX by Forte (31) was stabilized with fluoride as was the rat brain enzyme (20) which retained sensitivity to fluoride and was stabilized for 7 days at 4°C. Potent nucleotide phosphohydrolases were observed both in liver membranes and in the solubilized adenylate cyclase preparation. Under the condition of adenylate cyclase assay over 56% of the added nucleotide was hydrolyzed to the nucleoside. The ATP regenerating system used widely to maintain a substrate optimum for adenylate cyclase in membrane systems containing “ATPases” decreases the appearance of the dephosphorylated products of GTP, ITP, and GDP but not more than 15%.


Thus, it does not appear likely that a fourfold increase in adenylate cyclase activity due to GDP was due to a 16 and 60% conversion of GDP to GTP by membranes or the solubilized enzyme, respectively. The adenylate cyclase activation obtained with nucleotides cannot be due to sparing of substrate ATP from hydrolysis. The amount of substrate remaining after assay was, within the limits of the chromatographic methods employed, the same whether or not activation was observed. Thus, 20% to fivefold activation of adenylate cyclase was observed which was concomitant with equivalent substrate concentrations after assay. At low ATP concentrations, added nucleotide triphosphates spare ATP from hydrolysis up to 60% but this does not account for the effects of nucleotides on adenylate cyclase activation. Notwithstanding similar rates of hydrolysis of GTP, ITP, and GMP-PCP by solubilized membranes (Table V) the degree of activation of the soluble adenylate cyclase by these nucleotides was 40, 101, and 180%, respectively, when compared at 0.1 mM nucleotide (Fig. 1). In addition, the effects of GMP-PCP and GMP-PNP (Table VII), nonphosphorylating analogs of GTP, and of nucleoside diand monophosphates (Table IV) mitigate against the possibility that the nucleotides exert their action by a sparing effect on substrate ATP. GMP-PNP stimulated adenylate cyclase activity in membranes isolated from several different sources (29, 31) including rat liver (29). In the rat liver membrane system (29) GMP-PNP was 17 times as potent as GTP which increased basal activity by 20%. As reported here the solubilized enzyme prepared from rat liver membranes was equally sensitive to GMP-PCP and GMP-PNP both of which doubled basal activity. On the basis of their findings Londos et al. (29) have suggested that since GMP-PCP and GMP-PNP are not readily cleaved the guanyl nucleotides act as allosteric regulators of the adenylate cyclase system and not as phosphate donors. In our experiments, however, as much as 25% of GMP-PCP was hydrolyzed to 5’-GMP. We have also observed the hydrolysis of GMP-



PNP (Unpublished, N. I. Swislocki and J. Tierney). In adipocytes (331, and adipocyte membranes (15), thyroid membranes (101, as well as liver plasma membranes (341, inhibitory effects of GTP on adenylate cyclase have been observed. Harwood et al. (15) have proposed that phosphorylation of the enzyme can account for its inhibited state. The regulatory effects of guanyl nucleotides are complex and it is not clear at this juncture which nucleotide derivative is the effective agent nor what is the mechanism of guanyl nucleotides in regulating adenylate cyclase activity. In the course of studies of the interaction of guanyl nucleotides with isolated liver plasma membranes we demonstrated that [lIC]GTP or a hydrolytic product thereof bound to membranes (17). We have also observed (Unpublished, N. I. Swislocki and J. Tierney) the association of a partially purified adenylate cyclase preparation derived from Lubrolsolubilized membranes with [3H]GMPPCP or a hydrolytic product during the course of which 18% of added nucleotide was hydrolyzed (cf. Table V), to 5’-GMP. The association of [3H]GTP with a detergent-free dog heart adenylate cyclase has also been demonstrated (35). It is probably premature to assign a primary regulatory role (3-5, 7-9, 12, 13, 16, 18) to GTP to the exclusion of its hydrolytic products since the hydrolytic products themselves are effective in modulating adenylate cyclase activity obtained from several sources. Moreover, the examination of the effects of guanyl nucleotides made in membrane systems which are likely to possess nucleotide phosphohydrolases similar to those reported by us earlier (17) and examined more fully in this report have not been accompanied by direct evaluation of guanyl nucleotide hydrolysis. In view of the present observation of extensive hydrolysis of guanyl nucleotides both by membranes and by solubilized membranes an analysis of a role for nucleotides in regulating adenylate cyclase activity must await isolation of nucleotide phosphohydrolase-free nucleotide sensitive adenylate cyclase preparations. It may be,



however, that nucleotide hydrolysis is related to mechanism of adenylate cyclase control by guanyl nucleotides. Note added in proof. After this manuscript was submitted we isolated highly purified adenylate cyclase by direct affinity chromatography, on agarosehexane-GTP (30), of Lubrol-solubilized liver plasma membranes. The enzyme eluted from the affinity resin was homogenous on acrylamide gels and was devoid of phosphohydrolase activity found in the parent preparation. We could not observe activation of the purified adenylate cyciase by 0.1 mM GMP-PNP. REFERENCES 1 SUTHERLAND, E. W., @YE, I.. AND BUTCHER, R. W. (1965) Recent Progr. Hormone Res. 21, 623-642. 2. RODBELL, M., BIRNBAUMER. L., POHL. S. L.. AND KRANS, H. M. J. (1971) J. Biol. Chem. 246, 1877-1882. 3. LERAY, F., CHAMBAUT, A. M.. AND HANOC’NE. J. (1972) Biochem. Biophys. Res. Commun. 48, 138551391. 4. LERAY. F.. CHAMBAL-T, A. M., PERRENOUD. M. L.. A&D HANOUNE, J. (1973) Eur. J. Biochem. 38, 185-192. 5. GOLDFINE. I. D., ROTH, J.. AND BIRNBAUMER, L. (1972) J. Biol. Chem. 247, 1211-1218. 6. Kuo, W. N., HOGKINS, D. S., AND Kuo. J. F. (1973) J. Biol. Chem. 248, 2705-2711. 7. JOHNSON, D. G., THOMPSON, W. J.. AND WILLIAMS. R. H. (1973) Riochemistrv 13, 1920-1924. 8. BOCKAERT,J.. ROY, C.. AND JARD. S. (1972) J. Riol. Chem. 247, 7073-7081. 9. KRISHNA, G.. HARWOOD, J. P., BARBER, A. J.. AND JAMIESON, G. A. (1952) J. Biol. Chem. 247, 2253-2254. 10. WOLFF, J.. AND COOK. G. H. (1973) J. Biol. Chem. 248, 350-355. 11. SATO, S., YAMADA. T., TURIKATA, R., AND MAKIUCHI, M. (1974) Biochim. Biophys. Acta 332, 166-174. 12. BILEZIKIAN, J. P., AND AURBACH, G. D. (1974) J. Biol. Chem. 249, 157-161. 13. SIEGEL. M. I.. AND CUATRECASAS.P. (1974) Mol. Clin. Endocrinol. 1, 89-98. 14. DALTON, C.. HOPE, H., AND SHEPPARD, H. (1974) Arch. Biochem. Biophys. 163, 238-245. 15. HARWOOD. J. P.. LOW, H., AND RODBEL.L.M. (1973) J. Biol. Chem. 248, 6239-6245. 16. RODBELL, M.. LIN. M. C.. AND SALOMON, Y. (1974) J. Biol. Chem. 249, 59-65. 17. SWISLOCKI. N. I., SCHEINBERC. S.. AND SONENBERG, M. (1973) Biochem. Riophvs. Res. Commun. 52, 313319. 18. SWISLOCKI, N. I.. SONENBERG. M.. POSTEL-VINAY. M-C.. GOIDL. J. A., ROBERTS,J. E.. HENDERSON,


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AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. SWISLOCKI, N. I. (1970) Anal. Biochem. 38, 260-269. RANDERATH, E., AND RANDERATH, K. (1964) J. Chromatogr. 16, 126-129. LONDOS, C., SALOMON, Y., LIN, M. C., HARWOOD, J. P., SCHRAMM, M., WOLFF, J., AND RODBELL, M. (1974) l’roc. Nat. Acad. Sci. USA 71, 3087-3090. SWISLOCKI, N. I., MAGNUSON, T., AND TIERNEY, J. (1975) Fed. Proc. 34, 694. LEFKOWITZ, R. J. (1974) J. Biol. Chem. 249, 6119-6124. FORTE, L. R. (1972) Biochim. Biophys. Acta 266, 524-542. CRYER, P. R., JARETT, L., AND KIPNIS, D. M. (1969) Biochim. Biophys. Acta 172, 586-590. SWISLOCKI, N. I., AND SCHEINBERG, S. (1973) Fed. Proc. 32, 555. LEFKOWITZ, R. J. (1974) Fed. Proc. 33, 1249.

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

ARCHIVES OF BIOCHEMISTRY Activation AND BIOPHYSICS 168, 455-462 (197.5) of Solubilized Liver Membrane by Guanyl Nucleotides’ NORBERT Memorial I...
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