Eur. J . Biochem. 78, 337-342 (1977)

Evidence for an Adenylate-Cyclase Activity in Neurosecretory Granule Membranes from Bovine Neurohypophysis Dominique BONNE, Pierre NICOLAS, Marc LAUBER, Maryse CAMIER, Andree TIXIER-VIDAL, and Paul COHEN

Groupe de Neurobiochimie Cellulaire et Moleculaire, Unite d’Enseignement et de Recherches de Biochimie, Universite Pierre-et-Marie Curie, Paris and Groupe de Neuroendocrinologie Cellulaire du College de France, Paris

(Received January 29/May 9, 1977)

Purified bovine neurosecretory granules and their corresponding membranes were prepared after fractionation and purification processes from bovine pituitaries. An adenylate cyclase activity was detected both in the granules (apparent K, = 0.5 mM) and the corresponding preparations of the membranes (apparent K,,, = 0.5 mM). This enzyme was activated by fluoride in a way markedly dependent on the concentration of this ion, and with a maximum for a concentration of F ~ = 3.5 mM. The cyclase activity was also significantly enhanced by GTP. The reaction rate showed a strong dependance on the molar ratio [Mg2+]/[ATP] with maximal velocity for 7. It is suggested that this activity might play an important role in the control and regulation of neurosecretion in the neurohypophysis.

Neurophysin, ocytocin and vasopressin are pro- duced in specialized nuclei of the hypothalamus [ 1,2]. The packaging of these neurosecretory products involves the formation of membrane-limited vesicles originating from the Golgi body [3 ~ 81. The arrange- ment of the neurophysin . hormone complexes inside the granule core is not known. Studies in vitro have shown the importance of the neurophysin quaternary structure [9] and of particular structural features of the protein [lo] and of the neurohypophyseal nona- peptide hormones in their mutual interactions [l 1 ,121. In addition, reconstitution experiments, together with direct measurements of lipid content, have strongly suggested that the so-called ‘native neurophysin’ in- side the granule is probably not a lipoprotein [13].

Excretion of these products is believed to occur by exocytosis, involving fusion between the granule membrane and the plasma membrane [14- 161. Besides its role in the physical maintenance of the neurosecretory compounds, the granule membrane may provide a control system for exchange processes and a locus for the reception and transmission of regulatory signals. Although biosynthetic evidence for the conversion of a putative precursor into

neurophysin during axonal transport was recently presented [17], little is known about the mechanism of the possible molecular rearrangements of these neurosecretory products. These may occur inside the granule core between the site of granule packaging and the exocytosis process. Ca2+ ions [18-201 and ATP [21] have been reported to participate in this release, but its mechanism is not yet clearly under- stood at the molecular level. In addition, protein phos- phorylation was also described in neurosecretory granules possibly involving a protein phosphokinase activated by cyclic AMP [22].

In view of these points and also of the molecular and structural relationships which seem to exist between the granule membrane and the plasma mem- brane, we have investigated the hypothesis that these vesicle membranes might possess an adenylate cyclase activity. The use of several steps of fractiona- tion together with the analysis of biochemical markers of the membranes and electron microscope observa- tions allow us to show that our preparations are rela- tively homogeneous and possess this adenylate cyclase activity. It is presumed that this enzyme activity might play a role in the neurosecretory processes.

Abbreviations. Cyclic AMP, adenosine 3’ : 5’-monophosphate; (Na’, K +, Mg2 ‘)-ATPase, sodium/potassium/magnesium-stimulat- ed adenosine triphosphatase; Mg2 +-ATPase, magnesium-stimulated adenosine triphosphatase; Ca2 +-ATPase, calcium-stimulated adenosine triphosphatase.

Enzymes. Adenylate cyclase (EC 4.6.1 . l) ; S‘-nucleotidase or 5’-ribonucleotide phosphohydrolase (EC 3.1.3.5); ATPase (EC 3.6.1.3).

EXPERIMENTAL PROCEDURE

Biological Material

For each preparation, usually 30 pituitary glands were removed from adult cattle of both sexes within the

338 Adenylate Cyclase of Neurosecretory Granules

hour following animal sacrifice. The subsequent operations were conducted according to the procedure of Dean and Hope [23] at 4 "C, except for the modifications detailed in the following paragraphs.

Purified Granule Preparation

A highly purified granule fraction was prepared involving differential and sucrose gradient centrifuga- tions. All the experiments were run at 4 "C. The refractive index of sucrose solutions was measured by an Abbe refractometer and the corresponding densi- ties were deduced from a standard curve established at 20 "C. All the gravity values reported are average ones. Sedimentations were carried out using a JA20 rotor on a Beckman J21 centrifuge at 1100 x g for 15 min (fraction I), then at 7000 x g for 15 rnin and 26000 x g for 15 min. The final sediment was consid- ered to be a crude granular fraction and was resuspend- ed in 12 ml of cold 0.3 M sucrose (fraction 11). This preparation was further purified by sedimentation on a discontinuous density gradient according to Dean and Hope [23]. In order to improve the purifica- tion, the volume of each layer of sucrose was increased by a factor of 8 and the run was conducted on a Beck- man SW-27 rotor at 130000 x g for 2 h at 2 "C. Two major bands appeared under these conditions and the lower layer (Q = 1.23 g/cm3) was recovered; this corresponds to fraction E of Dean and Hope [23] which, according to these authors, consists of neuro- secretory granules free from lysosomal or mito- chondrial contaminations [23 - 271.

Membrane Preparations

Neurosecretory Granule Membranes. The recovered fraction E was diluted in 5 mM Tris-HC1 buffer pH 7.80 to 50 ml and .gently stirred at 4 "C for 30 min. Sedimentation of this dispersion at 30000 x g for 20 rnin gave a pellet which was resuspended in 1 ml 5 mM Tris-HC1 buffer pH 7.80 (fraction M). Fraction M was subsequently relayered at the top of a con- tinuous sucrose density gradient (17 - 68 %) and cen- trifuged for 2 h at 120000 x g at 2 "C. The major band at e = 1.185 g/cm3 was recovered from the gradient, rediluted in 5 mM Tris-HC1 buffer pH 7.80 and then resedimented at 30 000 x g during 20 min. The sedi- ment, resuspended in 1 ml 5 mM Tris-HC1 buffer pH 7.80, gave fraction Mp. The proteins of the granule content were analyzed in the supernatant of fraction M by slab gel electrophoresis (10 % acryl- amide, 0.33 % bis-acrylamide, Tris/glycine pH 8.5) either in the absence or in the presence of 0.1 % sodium dodecyl sulfate. The gels were scanned on a Vernon photometer integrator. Bovine neurophysins I, I1 and C standards were prepared as previously described [28].

Plasma Membranes. These membranes were isolat- ed according to a modification of the method of Coleman et al. [29] as described by Vilhardt and Holmer [26]. Further purification was achieved using a continuous sucrose density gradient (17- 68 %). The principal band observed at Q = 1.135 g/cm3 was recovered, diluted in 5 mM Tris-HC1 buffer pH 7.80 and sedimented at 40 000 x g during 20 min. The pellet, resuspended in 1 ml of this buffer, constitued fraction P and was used as such for further analysis. Alternatively, these preparations could be kept frozen in liquid nitrogen without loss of the measured activities.

Enzyme Assays

A TPases were evaluated according to Vilhardt and Hope [30]. Inorganic phosphate was measured by the method of Swynghedauw et al. [31].

5 '-Nucleotidase was determined by the same tech- nique as for Mg2+-ATPase except that adenosine 5'-monophosphate was used as the substrate. All determinations were run in triplicate. Protein content was measured according to Lowry et al. [32] following NaOH digestion of the samples.

Adenylate Cyclase Activity. The conversion of [M-~~PIATP (NEN, Boston, 10 Ci/mmol) into cyclic AMP was evaluated according to a slight modifica- tion of the method of Salomon et al. [33]. The standard incubation mixture (0.1 ml) contained 50 mM Tris-HC1 pH 7.80, 50 mM MgC12, 5 mM [ce3'P]ATP (40 counts min-' pmol-I), 0.25 mM cyclic AMP, 100 U/ml creatine kinase, 7 mM phosphocreatine, 0.1 % bovine serum albumin and 10 - 200 pg of mem- brane protein. Reaction was stopped after 10 min at 37 "C, by addition of 0.5 ml of a solution containing 2 % sodium dodecylsulfate, 0.5 mM cyclic AMP, 0.33 mM ATP and 50 mM Tris-HC1 pH 7.80; cyclic [3H]AMP (approximately 2 . lo4 counts/min) in 50 pl was then added to monitor the cyclic AMP recovery during the subsequent steps. Each sample was then incubated for 10 min at 70 "C. Isolation of cyclic AMP was carried out according to method C of Salomon et al. [33], on a Dowex AG-50-WX-8 column and then on a neutral alumina column. Cyclic AMP was counted in a Unisolve (Sochibo) scintillation mixture after 12 h of equilibration on a Inter- technique SL-30 spectrometer. The results of each test, run in duplicate, were expressed as pmol cyclic AMP min-' (mg protein)-'.

Radioimmunoassays

The neurophysin content was measured by radio- immunoassay. A mixture of crude bovine neuro- physins I, 11 and C [28] was used both as a standard and as a tracer with rabbit antiserum against bovine neurophysins kindly provided by Dr J. J. Legros.

D. Bonne, P. Nicolas, M. Lauber, M. Camier, A. Tixier-Vidal, and P. Cohen 339

Table 1 , Purification of neurosecretory granules and of the corresponding membranes f rom bovine posthypophysis Enzyme activities and neurophysin concentration were determined as described in Methods on each fraction of'the successive steps of granule purification. n.d. = none detectable

Fraction ATPase activity 5'-Nucleo- Adenylate Neurophysins tidase cyclase

Na+,K+,Mg2+ M 2 ' CaZ +

pmol Pi (mg protein) ~' h-' pmol cyclic mg/mg protein AMP (mg protein)- ' min-'

I (homogenate) 2.6 11.5 9.5 4.2 49.7 0.34 P (crude plasmatic membranes) 57.7 100.2 115.1 77.8 300.0 n.d. I1 (crude granules) 24.4 32.0 28.0 6.4 93.8 0.54 E (purified granules) < 0.05 14.5 9.5 3.5 68.3 0.78

Mp (purifiedgranulemembranesfrom M) n.d. 54.5 41.6 n.d. 72.0 0.06 M (granule membranes from E) n.d. 13.4 10.7 2.1 25.2 0.20

This antiserum cross-reacted with the three bovine neurophysins. Each measurement was run in tri- plicate according to Robinson [34] on each fraction (Table 1).

Electron Microscopy

Fractions E (intact granules) or Mp (purified granule membranes) were diluted in 5 ml of cold 2 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 0.25 M sucrose. After 3 h at 4 "C, the frac- tions were then centrifuged at 30000 x g for 30 min. The fixative was then renewed and the final pellets were fixed again in the same tubes for 12 h at 4 "C. They were then rinsed in the same buffer for several hours, post-fixed in 2 % osmic acid in the same buffer for 1 h at 4 "C, and then treated for 30 min at room temperature in 2 % uranyl acetate in distilled water. They were dehydrated with ethanol and flat- imbedded in araldite. Thin sections were cut in a plane perpendicular to the pellet in order to verify the homogeneity of the material from top to bottom. They were stained by uranyl acetate and lead citrate.

Fraction P (plasma membrane) was centrifuged at 25000 x g for 20 min and the final pellet was fixed in the same tube by 2 % osmic acid in the same way as pellets E and Mp.

RESULTS

The fractionation procedure led to purified granule membranes apparently devoid of plasma membrane contamination. This was shown by the biochemical analysis of some enzyme activities used as markers of plasma membranes and by electron microscope examination. The purified granule fraction (E, Table 1) contained some residual (Na+, K', Mg2 +)-ATPase activity and also a significant amount of 5'-nucleo-

tidase activity (Table 1). The granule content was identified after osmotic shock of the vesicles, in the supernatant fraction, by slab gel electrophoresis. The three main species of bovine neurophysins (I, IT and C) were clearly detected by comparison with purified protein standards [28]. The radioimmunoassay per- formed on the supernatant fraction indicated that about 80 % of the granule content liberated after osmotic shock of the vesicles is constituted by immuno- reactive neurophysin-like material. This is corroborat- ed by the finding that, on gel electrophoresis, roughly 80 % of the total protein detected in the vesicle content could be assimilated to neurophysins according to their electrophoretic patterns.

The granule membrane fraction (M, Table l), ob- tained after osmotic shock of the vesicles, contained no trace of (Na', K', Mg2+)-ATPase but some residual 5'-nucleotidase activity. This was eliminated by further purification on a continuous density gradient. The resulting fraction Mp (Table l), devoid of the (Na', K+, Mg2 +)-ATPase and the nucleotidase activities, exhibited the Mg2 +-ATPase and Ca2 +-ATPase activ- ities already described [35,36].

At the electron microscope level (Fig. 1) the granule fraction E was devoid of plasma membrane contamina- tion. Profiles of long sheets of plasma membrane were found only in fraction P. The granule fraction consisted mainly of granules bound by a smooth membrane and with a mean diameter of 150 nm (100-200 nm) (Fig. 1 a). They were generally rounded but some of them were elongated and irregular in shape. Their content displayed various degrees of electron density. Very few of them appeared empty. In addition to the granules a few other structures such as empty mito- chondria, empty granules, rough microsomes and a few polysomes were apparent. The purified granule membrane fraction (Fig. 1 b) was mainly composed of empty smooth vesicles which displayed an irregular shape and the same size range as the secretory

340 Adenylate Cyclase of Neurosecretory Granules

Fig. 1. Electron micrograph of ( a ) a sample o j the granule pellet and ( b ) the granule membrane pellet. In (a) note the presence of the granule membrane (+) and the various shapes and electron density of the neurosecretory granules. Very few smooth vesicles are associated with the granules (2). Magnification x 30000. In (b) granule membranes (+) are identified by their shape and their size range similar to those ofneuro- secretory granules. They are associated with some rough microsomes and polysomes (2). Very few non-extracted granules are left. Magnifica- tion x 30000

granules. This fraction was contaminated by a few empty mitochondria and some rough microsomes.

Transformation of ATP in cyclic AMP was repeatedly detected in the granules (fraction E, Table 1) and also in the corresponding membrane preparations (M and Mp in Table 1) in a series of ten different experiments. Purification of the crude granules (frac- tion 11) resulting in fraction E (Table 1) led to a de- crease in the adenylate cyclase activity. This was due to the removal of the plasma membrane contamina- tion. Preparation of the granule membrane fraction M was accompanied by a 60% loss of cyclase activity (Table l), reflecting a partial denaturation probably consecutive to the osmotic shock of the granules. This cyclase activity was found to increase with the purification of the membrane from 25.2 to 70.0 pmol cyclic AMP . min-' . (mg protein)-' (Table 1). Mea- surements of the apparent K , of the enzyme on the granules indicated a value of 0.5 mM for ATP (mean of three experiments), with a maximum velocity equal to 70 pmolcyclic AMP . min-' . (mgproteh-', when the [Mg2+]/[ATP] molar ratio was 10 (Fig.2). The purified Mp fraction prepared from the mem- branes showed a similar activity with a comparable apparent K,. These nucleotide cyclases are known to be dependent upon the [Mg2+]/[ATP] molar ratio of the system [37]. A similar behaviour was observed in the case of the granule membrane adenylate cyclase (Fig.3). The maximum activity was detected for a molar ratio [Mg2+]/[ATP] equal to 7, compared with the maximum recorded at 10 in the case of the crude plasma membrane preparation (result not shown).

I " " ' " " '

B

Fig. 2. Adenylate cyclase activity of ( A ) purifiedgranules (fraction E) and ( B ) of purified granule membranes (fraction M p ) as a function of ATP concentration. All tests were run according to the standard conditions and contained 100 pg of granule proteins (A) or 10 pg of purified granule membrane proteins (B). The molar ratio [Mg*+]/[ATP] was 10

D. Bonne, P. Nicolas, M. Lauber, M. Camier, A. Tixier-Vidal. and P. Cohen 341

0 5 10 [Mg2']/[ATP] ( rnol i rnol)

Fig. 3. Adenylate cyclase activity o j purified granule membranes (fraction M,) us afunction o f the molar ratio [ M g Z ' ] / [ A T P J . All tests were done according to the standard conditions, except that ATP was 0.2 mM. Results are expressed as a percentage of the activity at a ratio [MgZ+]/[ATP] of 10 (20 pmol CAMP. min-' . mg protein-')

Fig.4. Adenylatr eycluse activity of granules (A) and of purified granule memhranes (a) a.r a function of [ F - J . Tests were done according to the standard conditions and contained 200 pg protein1 test for granules and 68 pg protein/test for purified granule membranes

This adenylate cyclase was also studied with respect to its sensitivity to various activators. Both fluoride ions and GTP were found to increase the basal level of this enzyme in the granules (E) and in the membrane fractions M and Mp (Table 1). The maximal enhancement, i .e. 160% increase of the basal level, was recorded when the concentration of F- was equal to 3.5 mM at a [Mg2+]/[ATP] ratio of 10 (Fig. 4). The well-described activation of the nucleotide cyclase activity by GTP could also be reproduced on the granule membranes (fraction Mp). Enhancements of 40 or 75% were obtained respectively for 5 or 10mM ATP when the [Mg2+]/[ATP] ratio was equal to 10. Several substances were also tested for

their possible role as effectors of the cyclase. Under our conditions, no effect could be detected on the basal level of the enzyme in the presence of 1 pM neuro- physin, ocytocin, vasopressin, 17 /I-estradiol, nicotine or dopamine, either on granule membranes or on plasma membrane preparations.

DISCUSSION

These studies provide evidence for the existence of an adenylate cyclase activity in the preparations of neurosecretory granules from bovine pituitaries and in the corresponding purified membranes. That this activity could not be accounted for by a contamina- tion of plasma membranes was assessed by a number of observations suggesting the homogeneity of the granule and membrane preparations: (a) the total loss of two enzyme markers of plasma membranes, i.e. the (Mg2+, Na', K+)-ATPase and the 5'-nucleotidase, and (b) the electron microscope observations of the granule fraction which verified the absence of plasma mem- brane contamination in the granule pellet.

This was corroborated by the significant increase in adenylate cyclase activity following purification of fraction M to Mp. It should be noted that a minimum of 25 "/, contamination of granule membranes by the plasma membranes would be necessary to account for the measured cyclase activity in fraction Mp. Such a contamination would have been definitely visible in the electron microscope.

Because of the heterogenous structure of the post- pituitary, which is composed of neurosecretory axons, axon swellings, axon terminals and of pituicytes [38], the problem of the nature of contaminants in our granule fractions must be discussed. These contami- nants represented a low proportion of the granule pellet ; they consisted of altered mitochondria and of rough microsomes. In the case of the hepatocytes, Cheng and Farquhar [39] did not found any cyclase activity in the rough microsomes. From the electron microscope examination we cannot exclude a possible contamination of the granule membrane fraction by smooth microsomes or Golgi elements from the p h i - cytes. Nevertheless the disappearance of both the 5'-nucleotidase and (Mg2 +, Na+, K +)-ATPase activity upon purification of the granule membrane fraction concomitantly with the enrichment in cyclase activity are not in favor of the existence of significant amounts of Golgi elements in this fraction. Indeed, such enzymatic activities are known to be associated with the Golgi elements of the liver [40].

Our observations suggest that an adenylate cyclase activity is included in the membrane of the secretory granules. There are, at least to our knowledge, only two other recent mentions in the literature of the pres- ence of an adenylate cyclase activity in intracellular smooth membranes: in the Golgi-rich fraction from

342 D. Bonne, P. Nicolas, M. Lauber, M. Camier, A. Tixier-Vidal, and P. Cohen: Adenylate Cyclase of Neurosecretory Granules

the liver [39] and in membranes of the chromaffin granules of the adrenal medulla [41]. There is no evidence at this time that the adenylate cyclase of the neurosecretory granule membranes can be activated by effectors other than F- and GTP or that cyclic AMP production is directly coupled to the ATP- stimulated release of the neurosecretory content. The fact that this enzyme was not activated by 170-estradiol and nicotine, which are known to stimulate in vivo the release of the neurohypophyseal granule content [42], does not exclude a physiological role of the adenylate cyclase of neurosecretory granule membranes during the release process. These agents may have their receptor sites at the perikarya level. It cannot be excluded that some interrelations may exist between the elements of recognition of Ca2+ in these mem- branes (i.e. binding protein) [43] and this adenylate cyclase during the exocytosis process. The possible involvement of this cyclase in the control of the secretion of neurohypophyseal hormones is consistent with the recent report of an increase of cyclic AMP content of the rat neural lobe under physiological stimulation of the hormonal release [44].

We gratefully acknowledge the helpful discussions of Drs M. A. Devynk and C. Roy, the skilful assistance of Christine Fahy and Renee Picart and the gift of labelled compounds from the Commissariat d 1’Energie Atomiyue, Saclay. This work was sup- ported in part by funds from the (Inivc~.riiP P. c’t 21. Curie. the Centre Mutionul de la Recherche Scientijkpe (ERA 693 and RCP 220), the Ddldgation G&nirak d la Recherche Scientifique et Technique and the Fondarion pour h Recherche MPdicale Franquise.

REFERENCES 1. 2. 3.

4.

5.

6.

7.

8.

9.

10.

Bargmann, W. & Scharrer, E. (1951) Am. Sci. 39, 255-259. Sachs, H. (1969) A h . Enzymol. 32, 327-372. Fawcett, C. P., Powell, A. & Sachs, H. (1968) Endocrinology,

Sachs, H., Fawcett, C. P., Takabatake, Y. & Portanova, R. (1969) Rec. Progr. Horm. Res. 25, 447-491.

Sachs, H., Saito, S. & Sunde, D. (1971) Mem. Soc. Endocrinol. 19,325 - 336.

Pickering, B. T., Jones, C. W. & Burford, G. D. (1971) in Neuroh,vpophysiul Hormones (Wolstenholme, G. E. W. & Birch, eds) No. 39, Ciba Foundation Study.

Sachs. H. & Takabatake, Y. (1964) Endocrinology, 75, 943- 948.

Takabatake, Y. & Sachs, H. (1964) Endocrinolozy, 75, 934- 942.

Nicolas, P., Camier, M., Dessen, P. & Cohen, P. (1976) J . Biol. Chem. 251, 3965-3971.

Wolff, J., Alazard, R., Camier, M., Griffin, J. H. & Cohen, P. (1975) J . Bid . Chem. 250, 5215-5220.

83, 1299-1310.

11. Cohen, P., Camier, M., Wolff, J., Alazard, R., Cohen, J. S. &

12. Convert, O., Griffin, J. H., Di Bello, C., Nicolas, P. & Cohen,

13. Camier, M., Nicolas, P. & Cohen, P. (1976) FEBS Lett. 67,

14. Cheng, K . W. & Friesen, H . G. (1970) Metaholi.~m, 19, 876. 15. Douglas, W. W. (1968) Br. J . Pharmacol. 34, 451-474. 16. Douglas, W. W. & Nagasawa, J. (1971) J . Physiol. (L0nd.J

17. Gainer, H.. Sarne. Y. & Brownstein, M. J . (1977) Science

Griffin, J. H. (1975) Ann. N. Y. Acad. Sci. 248, 463-479.

P. (1977) Biochemistry, in the press.

137- 142.

218,94 P.

38.

19.

20.

21.

22. 23. 24.

25. 26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

37.

38. 39. 40.

41.

42. 43.

44.

(Wash. D. C. ) 195, 1354-1356.

172, 1 - 18.

(Lond.) 23/,96-98P.

479- 481.

531-540.

Douglas, W. W. & Poisner, A. M. (1964) J . Physiol. (L0nd.J

Dreifuss, J. J., Grau, J. D. & Nordmdnn, J. J. (1973) J . Physiol.

Nakazato, Y. & Douglas, W. W. (1974) Nature (Lond.) 24Y,

Poisner, A. M. & Douglas, W. W. (1968) M a / . Pharmacol. 4,

McKelvy, J. F. (1975) Ann. N . Y . Acad. Sci. 248, 80-89. Dean, C. R. & Hope, D. B. (1968) Biochem. J . 106, 565-573. Hope, D. B. & Pickup, J. C. (1974) Adv. Enzymol., 31, 403-

Pickup, J. C. & Hope, D. B. (1971) Biochem. J . 123, 153-162. Vilhardt, H. & Holmer, E. (1972) Acta Endocrinol. 71, 638-

Vilhardt, H. & Tondevold. E. (1972) Acta Endocrinol. 70,

Camier, M., Alazard, R. & Cohen, P. (1973) Eur. J . Biochem.

Coleman, R., Michell, R. H., Finean, J. B. & Hawthorne, J. N.

Vilhardt, H. & Hope, D. B. (1974) Biochem. J . 143, 181 - 190. Swynghedauw, B.. Bouveret, P. & Piguet, V. (1970) Ann. Biol.

Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem.

Robinson, A,. Zimmerman. A,. Engleman, E. G. & Frants,

Poisner, A. M. & Douglas. W. W. (1968) Science (Wmh. D. C . )

Pickup, J. C . , Johnston, C. I., Nakamura, S., Uttenthal, L. 0.

Birnbaumer, L., Pohl. S. L. & Rodbell, M. (1969) J . Bid.

Nordmann, J. J. (1977) J . Anal. 123, 213-218. Cheng, H. & Farquhar, M. G. (1976) J . Cell. Biol. 70,660- 670. Bergeron, J. J. M., Ehrenreich, J. H., Siekevitz, P. & Palade, G.

Zinder, O., Nikodijevic, O., Hoffman, P. G. & Pollard, H. B.

Robinson, A. G. (1975) Ann. N . Y. Acad. Sci. 248, 246-254. Brastrom, C. O., Huang, Y . C., Breckcnridge. B. Mc. L. &

Wolf, D. J. (1975) PFOC. Nut1 Acad. Sci. U.S .A . 72, 64-68. Ruoff, H. J., Mathison, R. & Lederis, K. (1976) Neuroendo-

crinology, 22, 18 -29.

410.

648.

625-635.

32,207 - 21 4.

(1967) Biochim. Biophys. Acta, 135, 573- 577.

Clin. 28, 159- 163.

(1951) J . Biol. Chem. 193. 265-270.

58,541 - 548.

A. G. (1971) Metaholi~m, 20, 1138- 1147.

160, 203 - 204.

& Hope, D. B. (1973) Biochem. J . 132, 361-371.

Chem. 244, 3368- 3476.

(1973) J . Cell. Biol. 59, 73-88.

(1976) J . Biol. Chem. 251, 2179-2181.

D. Bonne, P. Nicolas, M. Lauber, M. Camier, and P. Cohen*, Groupe de Neurobiochimie Cellulaire et Moleculaire, U.E.R. de Biochimie, Universite Pierre-et-Marie-Curie, 96 Boulevard Raspail, F-75006 Paris, France

A. Tixier-Vidal, Groupe de Neuroendocrinologie Cellulaire, ColEge de France, 11 Place Marcellin-Berthelot. F-75231 Paris-Cedex-05, France

* To whom correspondence should be addressed