Summary
A highly enriched fraction of plasma membranes from the bovine adrenal medulla has been isolated by differential and sucrose gradient centrifugation. The membranes were found to occur as 0.1–0.5μ diameter vesicles and to equilibrate at a density of 1.13–1.14 g/ml. This fraction was characterized by 4-fold elevated levels of adenylate cyclase and 20-fold elevated levels of 5′-nucleotidase. Secretory vesicle membranes, isolated by repeated hypotonie and hypertonic shocks of whole vesicles, were found to equilibrate between d = 1.08 and d = 1.12 on a sucrose density step gradient. These membranes were highly enriched in cytochrome b562 and dopamine-β-hydroxylase. Proteins in the two membranes were compared by SDS gel electrophoresis. All protein size classes found in the vesicle membrane fraction were also represented in the plasma membrane fraction, though in different proportions on the basis of staining intensity. The plasma membrane fraction contained prominent bands co-migrating with the α- and β-bands of tubulin, as well as a component co-migrating with actin. These bands were absent from the vesicle membranes. Fingerprint analysis of stained bands from the membrane fraction demonstrated that the components were indeed tubulin and actin. The plasma membranes contained twice as much sialic acid residues as did the chromaffin granule membranes, but had only half the cholesterol content on a weight basis. The cholesterol∶phospholipid ratio in the plasma membranes was 0.63, while in the secretory vesicle membranes it was 1.04. These results show that plasma membranes and secretory vesicle membranes are functionally and structurally different.
Similar content being viewed by others
References
Abrahams, S.J., Holtzman, E.: Secretion and endocytosis in insulin-stimulated rat adrenal medulla cells. J. Cell Biol. 56, 540–554 (1973)
Anton, A.H., Sayer, D.F.: A study of the factors affecting the aluminum oxidetrihydroxyindole procedure for the analysis of catecholamines. J. Pharmacal. exp. Ther. 138, 360–375 (1967)
Berl, S., Puszkin, W., Nicklas, J.: Actomyosin-like protein in brain. Science 179, 441–446 (1973)
Blitz, A.L., Fine, R.E.: Muscle-like contractile proteins and tubulin in synaptosomes. Proc. nat. Acad. Sci. (Wash.) 71, 4472–4476 (1974)
Bonne, D., Nicolas, P., Lauber, M., Camier, M., Tixier-Vidal, A., Cohen, P.: Evidence for an adenylate cyclase activity in neurosecretory granule membranes from bovine neurohypophysis. Europ. J. Biochem., 18, 337–342 (1977)
Bonner, W.M., Pollard, H.B.: The presence of F3-F2al dimers and F1 oligomers in chromatin. Biophys. biochem. Res. Commun. 64, 282–288 (1975)
Burridge, K., Phillips, J.H.: Association of actin and myosin with secretory granule membranes. Nature (Lond.) 254, 526–529 (1975)
Creutz, C.E.: Isolation, characterization and localization of bovine adrenal medullary myosin. Cell Tiss. Res. 178, 17–38 (1977)
Cuzner, M., Davison, A.N.: The lipid composition of rat brain myelin and subcellular fraction development. Biochem. J. 106, 29–34 (1968)
]Dittmer, J.C., Wells, M.A.: Quantitative and qualitative analysis of lipids and lipid components. In: Methods in enzymology (P. Colowick and N.O. Kaplan, eds.), pp. 482–530. New York-London: Academic Press 1969
Douglas, W.W.: Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Brit. J. Pharmacol. 34, 451–474 (1968)
Eagles, P.A.M., Johnson, L.N., VanHorn, C.: The distribution of concanavalin A receptor sites on the membrane of chromaffin granules. J. Cell. Sci. 19, 33–54 (1975)
Fairbanks, G., Steck, T.L., Wallach, D.F.H.: Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochem. 10, 2606–2617 (1971)
Fishman, W.H.: β-glucuronidase. In: Methods of enzymatic analysis (H.-U. Bergmeyer, ed.), pp. 869–874. New York-London: Academic Press 1965
Flatmark, T., Lagercrantz, H., Terland, O., Helle, K.B., Stjarne, L.: Electron carriers of the bovine adrenal chromaffin granules. Biochim. biophys. Acta (Amst.) 245, 252–262 (1971)
Grynszpan-Winograd, O.: Morphological aspects of exocytosis in the adrenal medulla. Phil. Trans. B 261, 291–292 (1971)
Heuser, J.E., Reese, T.S.: Evidence for the recycling of synaptic vesicle membranes during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57, 315–344 (1973)
Hirano, H., Parkhouse, B., Nicolson, G.L., Lennox, E.S., Singer, S.J.: Distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: its implications for membrane biogenesis. Proc. nat. Acad. Sci. (Wash.) 69, 2945–2949 (1972)
Hoffman, P.G., Zinder, O., Nikodijevic, O., Pollard, H.B.: ATP-stimulated transmitter release and cyclic AMP synthesis in isolated chromaffin granules. J. Supramol. Structure 4, 181–184 (1976)
Hurlbut, W.P., Ceccarelli, E.: Transmitter release and recycling of synaptic vesicle membrane at the neuromuscular junction. In: Advances in cytopharmacology (B. Ceccarelli, F. Clementi and J. Meldolesi, ed.), pp. 141–154. New York: Raven Press 1976
Izumi, F., Oka, M., Morita, K., Azuma H.: Catecholamine releasing factor in bovine adrenal medulla. FEBS Lett. 56, 73–76 (1975)
Jamieson, J., Palade, G.: Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic cells. J. Cell Biol. 50, 135–158 (1971)
Kates, M.: Techniques in lipid methodology. Part II of laboratory techniques in biochemistry and molecular biology (T.S. Work and E. Work, ed.), pp. 267–601. New York: American-Elsevier Publishing Co., Inc. 1971
Kirshner, N., Sage, H.J., Smith, W.J.: Mechanism of secretion from the adrenal medulla. 2. Release of catecholamines and storage vesicle protein in response to chemical stimulation. Molec. Pharmacol. 3, 254–265 (1967)
Korn, E.D.: Cell membranes: structure and synthesis. Ann. Rev. Biochem. 38, 263–288 (1969)
Laemmli, V.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680–685 (1970)
Larsen, C., Dallner, G. Ernster, L.: Association of sialic acid with microsomal membrane structures in rat liver. Biochem. biophys. Res. Commun. 49, 1300–1306 (1972)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Mann, G.V.: A method for measurement of cholesterol in blood serum. Clin. Chem. 7, 275–284 (1961)
McCaman, R.E., McCaman, M.W., Hunt, J.M., Smith, M.S.: Microdetermination of monoamine oxidase and 5′-hydroxytryptophan decarboxylase activities in nervous tissues. J. Neurochem. 12, 15–23 (1965)
Meyer, D.I., Burger, M.M.: The chromaffin granule surface. Localization of carbohydrate on the cytoplasmic surface of an intracellular organelle. Biochim. biophys. Acta (Amst.) 443, 428–436 (1976)
Michell, R.H., Hawthorne, I.W.: The site of diphosphoinositide synthesis in rat liver. Biochem. biophys. Res. Commun. 21, 333–338 (1965)
Mollinoff, P.B., Winshilboum, R., Axelrod, J.: A sensitive enzymatic assay for dopamine-β-hydroxylase. J. Pharmacol. exp. Ther. 178, 425–431 (1971)
Nahas, G.G., Zagury, D., Milhand, A., Manger, W.M., Pappas, G.: Academia and catecholamine output of the isolated canine adrenal gland. Amer. J. Physiol. 213, 1186–1192 (1967)
Nelson, G.J.: Composition of neutral lipids from erythrocytes of common mammals. J. Lipid Res. 8, 374–379 (1967)
Nijjar, M.S., Hawthorne, J.N.: A plasma membrane fraction from bovine adrenal medulla: preparation, marker enzyme studies and phospholipid composition. Biochim. biophys. Acta (Amst.) 367 (2), 190–201 (1974)
Nikodijevic, O., Nikodijevic, B., Zinder, O., Yu, M-Y.W., Guroff, G., Pollard, H.B.: Control of adenylate cyclase from secretory vesicle membranes by β-adrenergic agents and nerve growth factor. Proc. nat. Acad. Sci. (Wash.) 73, 771–774 (1976)
Ora, L., Amherdt, M., Malaisse-Lagal, F., Rouiller, C., Renold, A.E.: Insulin release by emiocytosis: demonstration with freeze-etching technique. Science 179, 82–84 (1973)
Phillips, J.M., Slater, A.: Actin in the adrenal medulla. FEBS Lett. 56, 327–331 (1975)
Poisner, A.M.: In: Frontiers in neuroendocrinology (W.F. Ganong and L. Martini, ed.), pp. 33–39. New York: Oxford University Press 1973
Pollard, H.B., Miller, A., Cox, G.C.: Synaptic vesicles: structure of chromaffin granule membranes. J. Supramol. Structure 2, 295–306 (1973)
Pollard, H.B., Zinder, O., Hoffman, P.G.: In: The cell surface. Immunological and chemical approaches (B.D. Kahan and R.A. Reisfeld, ed.), pp. 64–79. New York: Raven Press, 1974
Pollard, H.B., Zinder, O., Hoffman, P.G., Nikodijevic, O.: Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogues and external pH. J. biol. Chem. 251, 4544–4550 (1976)
Pollard, T.M., Korn, E.D.: Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J. biol. Chem. 248, 448–450 (1973)
Rambourg, A., Leblond, C.P.: Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat. J. Cell Biol. 32, 27–53 (1967)
Salomon, Y., Londos, C., Rodbell, M.: A highly sensitive adenylate cyclase assay. Anal. Biochem. 58, 541–548 (1976)
Schneider, F.H., Smith, A.D., Winkler, H.: Secretion from the adrenal medulla: biochemical evidence for exocytosis. Brit. J. Pharmacol. 31, 94–104 (1967)
Schwartz, A., Nagano, K., Nakao, M., Lindenmayer, G.F., Allen, J.C.: The sodium and potassium activated adenosine triphosphatase system. In: Methods in pharmacology (A. Schwartz, ed.), pp. 368–371 New York: Appleton-Croft, 1971
Smith, A.D.: Biochemistry of adrenal chromaffin granules. In: The interaction of drugs and subcellular componets in animal cells (P.N. Campbell, ed.), pp. 239–292. London: Churchill Ltd. 1968
Smith, A.D., Winkler, H.: A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem. J. 103, 480–482 (1967)
Smith, U., Smith, D.C., Winkler, H., Ryan, J.W.: Exocytosis in the adrenal medulla demonstrated by freeze-etching. Science 179, 79–82 (1973)
Spudich, J.A.: Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. J. biol. Chem. 249, 6013–6020 (1974)
Stetten, M.R., Goldsmilh, P.K.: A Limulus glucose-6-phosphatase with phosphotransferase activity characteristic of vertebrate liver microsomes. Biochim. biophys. Acta (Amst.) 444, 835–852 (1976)
Taugner, G., Hasselbach, W.: Über den Mechanismus der Catecholamin-Speicherung in den “chromaffinen Granula” des Nebennierenmarks. Naunyn-Schmiedebergs Arch. Pharmakol. exp. Path. 255, 266–286 (1966)
Virtaneu, I., Wantiovaava, J.: Visualization of charged groups on the surface of rat liver nuclei. J.Cell Sci. 16, 665–676 (1974)
Wilson, S.P., Kirshner, N.: Isolation and characterization of plasma membranes from the adrenal medulla. J. Neurochem. 27, 1289–1298 (1976)
Author information
Authors and Affiliations
Additional information
Supported, in part, by a stipend to O.Z. from The Grant Foundation, New York
Rights and permissions
About this article
Cite this article
Zinder, O., Hoffman, P.G., Bonner, W.M. et al. Comparison of chemical properties of purified plasma membranes and secretory vesicle membranes from the bovine adrenal medulla. Cell Tissue Res. 188, 153–170 (1978). https://doi.org/10.1007/BF00222628
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00222628