Abstract. Permeabilized adrenal chrornaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations . Recently, we have demonstrated that chrornaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T

Abstract. Annexin II is a Ca2+-dependent membranebinding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, pll. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked cornpletely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K ÷. The light chain of annexin II, pll, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells, pll can modify the Ca 2÷-and/or the phospholipid-binding properties of p36. We found that less Ca 2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of pll in adrenergic cells may confer a higher Ca 2+ affinity to the exocytotic pathway in these cells.

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

Abstract. The subcellular distribution of the 43,000-D protein (43 kD or u~) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghirm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool.Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R.J., and Z. W. Hall, 1983, J. CellBiol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry.The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and noninnervated domains of the plasma membrane.T HE postsynaptic membrane of the neuromuscular junction and of the electromotor synapse corresponds to a local differentiation of the plasma membrane characterized by an accumulation of the nicotinic acetylcholine receptor (Ach-R).t This membrane specialization persists for a long period after denervation (reviewed in references 10 and 18), indicating that physical constraints maintain the Ach-R molecules in place and, in particular, prevent against their lateral diffusion. Interactions of the Ach-R with extrinsic components from the extracellular matrix (13, 55) and/or with the cytoskeleton (17, 48, 67) have been postulated to contribute to this differentiation process. cell--the non-innervated face--is specialized in the regeneration of the electrochemical gradient (40). The Ach-R is present exclusively on the innervated membrane, and Na ÷ K ÷ ATPase accumulates on the non-innervated one. These membranes thus constitute fully differentiated and stable domains of the plasma membrane. From this standpoint the diskshaped electrocyte shows a striking functional and structural polarity ( 18, 21).Postsynaptic membrane fractions purified from Torpedo comprise essentially the intrinsic Ach-R polypeptides plus few extrinsic components of apparent molecular mass 43 kD (63, 64) na...

Anions and particularly sulfate are known to interact with 3-phosphoglycerate kinase and to induce an increase of its catalytic efficiency. The present work affords information on the location of the anionic site and on the conformational change produced by the sulfate binding. We have established that sulfate is able, first, to modify the environment of some critical amino acids (cysteine and arginines) located in the N-terminal half of the protein, second, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm. All these modifications are produced by sulfate concentrations required for the activation of the enzyme. The most striking result consists in a large change in the hydrodynamic properties of the protein upon sulfate interaction as determined by analytical ultracentrifugation studies. Thus, sulfate modifies the shape of the molecular, causing it to become more compact. Furthermore, a study of the binary and ternary complexes between yeast 3-phosphoglycerate kinase and its substrates suggests that such a change of the shape of the molecular only occurs in sulfate-enzyme with or without substrates and in ATP (with or without Mg2+)-3-phosphoglycerate-enzyme complexes.

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We have previously demonstrated that brain spectrin binds to the low-molecular-mass subunit of neurofilaments (NF-L) [Frappier, Regnouf & Pradel (1987) Eur. J. Biochem. 169, 651-657]. In the present study, we seek to locate their respective binding domains. In the first part we demonstrate that brain spectrin binds to a 20 kDa domain of NF-L. This domain is part of the rod domain of neurofilaments and plays a role in the polymerization process. However, the polymerization state does not seem to have any influence on the interaction. In the second part, we provide evidence that NF-L binds to the beta-subunit of not only brain spectrin but also human and avian erythrocyte spectrins. The microtubule-associated protein, MAP2, which has also been shown to bind to microfilaments and neurofilaments, binds to the same domain of NF-L as spectrin does. Finally, among the tryptic peptides of brain spectrin, we show that some peptides of low molecular mass (35, 25, 20 and 18 kDa) co-sediment with either NF-L or F-actin.

Heterotetrameric annexin 2 phosphorylated "in vitro"by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at M calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.Annexin 2 is a calcium-phospholipid-binding protein of the annexin family which has been characterized in the adrenal medulla as chromobindin 8. The heterotetrameric molecule formed of two heavy chains of 36 kDa and two light chains of 11 kDa possesses the unique property among the other annexins to aggregate chromaffin granules at micromolar calcium concentration (1).It has been shown by immunoelectron microscopy (2) that in chromaffin cells annexin 2 was closely associated with the inner face of the plasma membranes. In cultured chromaffin cells, thin strands were found cross-linking the chromaffin vesicles to the plasma membrane after stimulation with acetylcholine. Similar thin strands were also observed between aggregated chromaffin vesicles when they were mixed with annexin 2 in the presence of calcium. These data strongly suggested that conformational changes were induced in annexin 2 to cross-link the vesicles and the plasma membrane after stimulation of cultured chromaffin cells. When primary cultured chromaffin cells were stimulated by nicotine, annexin 2 was phosphorylated by protein kinase C. The phosphorylation of the protein was concomitant with the catecholamine release.1 In streptolysin-permeabilized cells, annexin 2 phosphorylated "in vitro" by brain protein kinase C was able to reconstitute secretion of catecholamines in cells depleted of protein kinase C activity (3). Taken together, all these results suggest that annexin 2 could be involved in the exocytotic process....