Bovine Adrenal Medullary Chromaffin Granules Research Articles

Characterization and location of post-translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules.

Bovine chromoganin B (CGB)/secretogranin I, an acidic protein with a sequence of 626 residues and an isoelectric point of 5.2 is a major member of the chromogranin/secretogranin (CG/Sg) family. The difference between the theoretical molecular mass (76 kDa) and the value estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis results from post-translational modifications (glycosylation, phosphorylation and sulfation) and from the abundance of acidic residues (D 4.6%, and E 16.5%). Although the sequence of CGB is known, the structural analyses of the post-translational modifications have so far not been carried out. In the present study, using a combination of proteomic techniques including two-dimensional gel electrophoresis, Western blot, high-performance liquid chromatography purification, enzymatic digestion, sequencing, carbohydrate analysis, matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry analysis, we have located 18 post-translational modifications on bovine CGB, isolated from adrenal medulla chromaffin granules. Furthermore, we have identified at the molecular level the presence of a mutation M/V on position 577 of natural CGB. All together these data reflect the complex structure of this protein marker of the neuroendocrine system.

Antibacterial Activity of Glycosylated and Phosphorylated Chromogranin A-derived Peptide 173-194 from Bovine Adrenal Medullary Chromaffin Granules

Recently, we have isolated from bovine chromaffin granules and identified two natural peptides possessing antibacterial activity: secretolytin (chromogranin B 614-626) and enkelytin (proenkephalin-A 209-237). Here, we characterize a large natural fragment, corresponding to chromogranin A 79-431, that inhibits growth of both Gram-positive and Gram-negative bacteria. The aim of the present work was to determine the shortest active peptide located in the 79-431 chromogranin A region. Three peptides, which shared the same 173-194 chromogranin A sequence (YPGPQAKEDSEGPSQGPASREK) but differed in post-translational modifications, including O-glycosylation and tyrosine phosphorylation, were isolated. A detailed study using microsequencing and mass spectrometry allowed us to correlate their antibacterial activity with these post-translational modifications. The chromogranin A precursor fragment (79-431) and the active glycosylated and phosphorylated peptides were, respectively, named prochromacin and chromacin (P, G, and PG for phosphorylated, glycosylated, and phosphorylated-glycosylated form).

Characterization of glycoprotein II from bovine adrenal medullary chromaffin granules. Identification of components representing the secretory vesicle counterparts of the lysosomal-associated membrane glycoproteins (lamp-1 and lamp-2)

Glycoprotein II (GpII) is a heterogenous glycoprotein isolated from the membranes of bovine chromaffin granules in the adrenal medulla. When viewed by two-dimensional electrophoresis this glycoprotein consists of two components, upper (GpIIa) and lower (GpIIb), with a molecular mass of 80,000-100,000 daltons and a pI of 4.2-4.7. NH2-terminal sequence analysis of GpIIa and GpIIb revealed sequence similarity with lysosomal membrane glycoproteins (lamp-1 and lamp-2), which was supported by sequence data of peptides from trypsin and cyanogen bromide digestions. An oligonucleotide probe was used to isolate a cDNA clone encoding the nucleotide sequence of GpIIa. The predicted amino acid sequence of GpIIa shares a 72% identity with the human lamp-1 type protein, which belongs to a highly conserved group of lysosomal-associated membrane glycoproteins (lamp proteins), whose function is still unknown. The COOH-terminal region of GpIIa was identical to the COOH-terminal region of lamp proteins. This COOH-terminal determinant has been demonstrated to be essential for the intracellular targeting of lamp proteins to lysosomes. A synthetic peptide antisera to the COOH-terminal region of GpIIa was used to show that this region is present on purified chromaffin granules and not proteolytically processed. The sequence analysis of GpIIa and immunological data confirm GpII as the secretory granule counterpart of lamp proteins and raise some questions regarding intracellular targeting between lysosomes and secretory granules within the chromaffin cell.

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process.

Synexin (annexin VII) is a Ca(2+)- and phospholipid-binding protein which has been proposed to play a role in Ca(2+)-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 micrograms/mg) and Ca2+ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/microns 2) and in the cytosol (5.3 particles/microns 2), but mainly around the granule membrane in the granular cell area (11.7 particles/microns 2). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.

Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis

We have characterized glycoprotein H (GpH) from bovine adrenal medullary chromaffin granules. Two-dimensional gel electrophoresis was used to purify GpH from an insoluble fraction obtained following extraction of chromaffin granule membranes with lithium diiodosalicylate. The GpH material was recovered from two-dimensional gel spots by concentration and recovery on a one-dimensional gel followed by electro-blotting to a poly(vinylidene difluoride) membrane. This material was subjected to in situ tryptic digestion. The released peptides were purified by microbore high performance liquid chromatography and sequenced. The peptide sequences revealed extensive similarity to the mammalian kex2/subtilisin-related proteases (PC2 and PC3) which have been characterized recently by molecular cloning and sequence analysis (Smeekens, S. P., and Steiner, D. F. (1990) J. Biol. Chem. 265, 2997-3000; Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J., and Steiner, D. F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 340-344). The sequence similarity included regions that contain residues equivalent to the aspartic acid and histidine residues which are involved in the active site of the subtilisin family of serine proteases. The sequence data revealed the presence of tryptic peptides derived from both PC2 and PC3. NH2-terminal sequence analysis of GpH gave two sequences which were aligned with residues 110-121 of PC2 and PC3. It is likely that these sequences represent the mature form of PC2 and PC3 in chromaffin granules. These forms would be generated by cleavage at a site which is conserved in mammalian kex2-related enzymes and which would result in the release of approximately 80-residue propeptides. It was concluded that the spot identified as GpH by two-dimensional gel electrophoresis contains the bovine counterparts of both PC2 and PC3. The direct identification of these components in chromaffin granules supports their role in the processing of protein precursors.

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Proteolytic processing enzymes are required to convert the enkephalin precursor to active opioid peptides. In this study, a novel 33-kDa thiol protease that cleaves complete precursor in the form of [35S]methionine preproenkephalin was purified from bovine adrenal medullary chromaffin granules. Chromatography on concanavalin A-Sepharose and Sephacryl S-200, chromatofocusing, and chromatography on thiopropyl-Sepharose resulted in an 88,000-fold purification with a recovery of 35% of enzyme activity. The thiol protease is a glycoprotein with a pI of 6.0. It cleaves [35S]methionine preproenkephalin with a pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0. Interestingly, production of trichloroacetic acid-soluble products was optimal at pH 4.0, suggesting that processing of initial precursor and intermediates may require slightly different pH conditions. The protease requires dithiothreitol for activity and is inhibited by the thiol protease inhibitors iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, and cystatin. These properties distinguish it from other thiol proteases (cathepsins B, H, L, N, and S), indicating that a unique thiol protease has been identified. The enzyme converted [35S]cysteine preproenkephalin (possessing [35S]cysteine residues specifically within the precursor's NH2-terminal segment) to 22.1-, 21.6-, 17.7-, 17.3-, and 15.0-kDa intermediates that contain the precursor's NH2-terminal segment; proenkephalin in vivo is converted to similar intermediates. The enzyme cleaves peptide F at Lys-Arg and Lys-Lys dibasic amino acid sites to generate methionine enkephalin and intermediates. The appropriate vesicular localization, pH optimum, proteolytic products, and cleavage site specificity suggest that this thiol protease may be involved in enkephalin precursor processing. Most interestingly, [35S]methionine beta-preprotachykinin, a precursor of substance P, is minimally cleaved, suggesting that the thiol protease may possess some selectivity for the enkephalin precursor.

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110–120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor protein in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla > anterior pituitary > pancreas > small intestine , hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.

The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.

Presence of pancreatic trypsin inhibitor in adrenal medullary chromaffin cells

A bovine pancreatic trypsin inhibitor was isolated from bovine adrenal medullary chromaffin granules. Its N-terminal sequence is: arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-gly-pro-cys-lys-ala-arg-ile-arg-tyr- phe-tyr-asn-ala-lys-ala-gly-leu-cys-gln-thr-phe-val-tyr-gly-gly-cys-arg-ala-lys- arg-asn-asn-phe-lys- which corresponds precisely with the N-terminus of Bovine Pancreatic Trypsin inhibitor. The presence of this inhibitor in these granules suggests another method of regulating the prohormone proteases present there.

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes: Purification and properties

Abstract A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.

An investigation of the molecular properties and stability of intermediates of proenkephalin in isolated bovine adrenal medullary chromaffin granules.

An antiserum to a synthetic peptide corresponding to residues 95-117 of bovine proenkephalin recognizes all the major intermediates of this prohormone in bovine adrenal medulla (Birch, N. P. and Christie, D. L. (1986) J. Biol. Chem. 261, 12213-12221). This antiserum enabled an investigation of the stability and molecular properties of intermediates in the processing of proenkephalin in bovine adrenal medullary chromaffin granules. Intact and hypotonic lysates of chromaffin granules were incubated at 37 degrees C and the stability of intermediates assessed by gel filtration followed by radioimmunoassay and gel electrophoresis in combination with immunoblotting. Processing was slow in intact granules compared with incubations of hypotonic lysates which resulted in the selective cleavage of an Mr 27,000 intermediate and increases in the amounts of immunoreactivity of lower molecular weight. Protease inhibitors increased the stability of the 27-kilodalton intermediate, the most effective being p-chloromercuribenzoate. Preliminary evidence was obtained for the regulation of the processing of this intermediate by soluble factors present in chromaffin granules. It appears that membrane-associated intermediates of proenkephalin are relatively stable, although analysis of soluble immunoreactivity released during the incubation of chromaffin granule membranes showed a decrease in the 27-kilodalton intermediate and increased amounts of lower molecular weight intermediates. Analysis of hypotonic lysates by two-dimensional gel-electrophoresis showed that proenkephalin intermediates exhibit significant microheterogeneity. It will be important to compare the products of proenkephalin generated by purified proteases with a putative role in the processing of this prohormone with the properties of endogenous intermediates as revealed in this study.

Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum.

An antiserum was generated against a synthetic peptide corresponding to amino acids 95-117 of bovine proenkephalin, and a sensitive radioimmunoassay was developed. Comparison of the reactivities of the synthetic peptide, its specific cleavage products, and other synthetic peptides showed that the important immunological determinant was contained within residues 101-109 of bovine proenkephalin (-Gly-Gly-Glu-Val-Leu-Gly-Lys-Arg-Tyr-). Radioimmunoassay of fractions after gel filtration of bovine adrenal medullary chromaffin granule lysate showed three pools of immunoreactivity: pool 1 (Mr 20,000-30,000), pool 2 (Mr 10,000-20,000), and pool 3 (Mr approximately 5,000). Further characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that the antiserum recognized 27-, 20.5-, 16.5-, and 5.6-kilodalton enkephalin-containing proteins. The radioimmunoassay was also used to detect proenkephalin-like material in extracts of rat adrenal and regions of rat brain and spinal cord following gel filtration. Immunoreactivity from the rat adrenal chromatographed predominantly as high molecular weight material (Mr 31,500-43,500), whereas material in regions of rat brain showed a broader molecular weight distribution (Mr 4,000-43,500). This indicated differences in the processing of proenkephalin between rat adrenal and brain tissue. Differences were also seen in the molecular weight profile of immunoreactivity in different brain regions, most noticeable in the case of striatum and hypothalamus, suggesting regional differences in processing. Based on quantitation of higher molecular weight immunoreactive proenkephalin-like material and free Met-enkephalin immunoreactivity in different brain regions, it was apparent that extensive processing of proenkephalin occurs in brain. We concluded that antisera against proenkephalin-(95-117) recognize a wide range of intermediates in the processing of proenkephalin in both bovine adrenal medulla and rat adrenal, brain, and spinal cord, making it a useful tool for further studies concerned with the expression and post-translational processing of proenkephalin.

Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal medullary chromaffin granule membranes by immunoblotting

An antiserum which recognizes high molecular mass enkephalin-containing proteins was used to compare proenkephalin intermediates in both the soluble and membrane components of bovine adrenal chromaffin granules by immunoblotting. While a range of molecular mass forms were identified in the soluble lysate the major form in the membranes corresponded to a 27-kDa enkephalin-containing protein. Enzymic digestion of bands of 27-kDa material and quantitation of the enkephalin released showed that 22% of this material was membrane-associated. High concentrations of chaotropic agents were required to extract this material from the membranes. Association of hormone and neuropeptide precursors with membrane components may by important for targeting of precursors to secretory granules or correct processing.

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

New bovine adrenal medullary peptide and its precursor.

We have isolated a previously unknown peptide and its precursor from bovine adrenal medullary chromaffin granules. The peptide sequence is Leu-Pro-Val-Asn-Ser-Pro-Met-Asn-Lys-Gly-Asn-Glu-Val-Met-Lys. The peptide is cleaved from the precursor at a Lys site. The sequence shows no homology to any known protein in the largest sequence data bank available.

Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine β-hydroxylase

Dopamine β-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose- N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine β-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine β-hydroxylase subunits of M r ≅ 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted α-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine β-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix.

The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes

The topological arrangement of cytochrome b 561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membranebound proteins. The labeled cytochrome b 561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b 561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.

The chromaffin granule as a model for membrane fusion: implications for exocytosis

Rapid freeze/freeze-fracture and thin section electron micrographic studies of the Ca 2+-promoted aggregation and fusion of isolated bovine adrenal medullary chromaffin granule membranes show that the granules undergo a series of morphological changes. The contact region becomes quite extensive and the membrane curvature changes radically at the edge of the contact site. The core material retracts away from the contact site leaving an electron lucent “stripe”; however, it remains adjacent to the membrane in the non-contact areas. The pentalaminar double membrane of the contact region often shows breaks. Close examination reveals that the two granule membranes have fused and become one continuous membrane. Rearrangement of large membrane associated particles (MAPs) can be seen by freeze fracture after Ca 2+-promoted granule-granule contact. The broken pentalaminar septum becomes smaller and may break down into globular structures. These observations suggest a series of reactions in which the granules first form an encounter complex, then a stable complex. The membranes within the contact region undergo lateral displacement of the proteins and phase separations of the lipids, and then fuse. Analysis of the kinetics of turbidity and fluorescence changes during granule aggregation and fusion support the main postulates of the model. The initial events of aggregation are facilitated by putative recognition proteins and K + will promote all activities except fusion. Recent observations that several soluble proteins (synexin, tubulin and albumin) will act as fusogens are discussed in terms of the relevance to exocytosis in vivo.

Catecholamine Uptake and Release in Isolated Chromaffin Granules Exposed to Halothane

The effects of halothane on the catecholamine release from chromaffin granules and the uptake of 14C-epinephrine into the granules were studied using isolated bovine adrenal medullary chromaffin granules. The granules were isolated with a Millipore filter, and were incubated for 10 min in an isotonic medium containing adenosine triphosphate (ATP)-Mg++. At halothane concentration of 0.4 mM or greater, the uptake of 14C-epinephrine into the granules was inhibited in a dose-related manner. At 0.7 mM halothane, epinephrine uptake was reduced to 56 per cent of control. The inhibition was completely reversible after removal of halothane from the medium. The inhibitory effect of halothane was not affected by high-osmolar medium. Catecholamine release from isolated chromaffin granules was enhanced by halothane at concentrations of more than 1.3 mM, but was not affected when the concentration was 0.7 mM or less. The results suggest the possiblity that halothane might alter the intracellular storage-turnover process and the subsequent release pattern of catecholamines. The possible mechanisms of this action are discussed.

Putative enkephalin precursors in bovine adrenal medulla

Extracts from bovine adrenal medulla and adrenal medullary chromaffin granules were found to contain three proteins, 20,000, 10,000 and 5,000 approximate molecular weights which yield tryptic peptides with opioid activity. The opioid activity of these peptides was demonstrated with a radioreceptor assay and two radioimmunoassays. The three proteins yield the same active peptides all of which are chromatographically distinct from the tryptic opioid nonapeptide β-LPH 61–69, generated by trypsin digestion of pituitary endorphins and their precursors. Furthermore, these endorphins and their precursors do not appear to be present in the adrenal medulla. These findings further support the hypothesis that the enkephalin biosynthetic pathway is distinct from that leading to β-endorphin.