Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.

In response to an external stimulus, neuronal cells release neurotransmitters from small synaptic vesicles and endocrine cells release secretory proteins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle membranes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense core granule membranes, monoclonal antibodies bound to beads were used to immunoisolate organelles containing the synaptic vesicle antigens. [3H]norepinephrine was used to label both chromaffin granules purified from the bovine adrenal medulla and rat pheochromocytoma (PC12) cells. Up to 80% of the vesicular [3H]norepinephrine was immunoisolated from both labeled purified bovine chromaffin granules and PC12 postnuclear supernatants. In PC12 cells transfected with DNA encoding human growth hormone, the hormone was packaged and released with norepinephrine. 90% of the sedimentable hormone was also immunoisolated by antibodies to all three proteins. Stimulated secretion of PC12 cells via depolarization with 50 mM KCl decreased the amount of [3H]norepinephrine or human growth hormone immunoisolated. Electron microscopy of the immunoisolated fractions revealed large (greater than 100 nm diameter) dense core vesicles adherent to the beads. Thus, large dense core vesicles containing secretory proteins possess all three of the known synaptic vesicle membrane proteins.

An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin.

A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.

Lysophospholipase activity of bovine adrenal medulla. A reevaluation.

1. Chromaffin granule preparations isolated from bovine adrenal medulla hydrolyzed endogenous lysophosphatidylcholine (lyso-PC) and generated lysophosphatidylethanolamine when dialyzed at pH 7.5. 2. Undialyzed granule preparations hydrolyzed exogenously added [1-14C]palmitoyl-lyso-PC maximally (12-16 nmol/min per mg) at pH 7.5. At a given concentration of protein, activity increased with increasing concentrations of substrate lyso-PC to a maximum beyond which substrate inhibited activity up to 95%. 3. More than 95% of the lysophospholipase activity of fresh granule preparations toward exogenously added lyso-PC was inactivated irreversibly by dialysis. 4. By contrast, fresh microsomal preparations from adrenal medulla had similar substrate requirements for maximal lysophospholipase activity, but more than 35% of the activity was retained at high substrate concentrations or after extensive dialysis. 5. We conclude that adrenal organelles, other than microsomes, contain potent membrane-associated lysophospholipase activity that is inactivated preferentially by high detergent-substrate concentrations and/or dialysis. These observations suggest that lysophospholipase activity (and perhaps phospholipase A activity) is more widely distributed in organelle membranes of the adrenal medulla than was reported previously.

31p nuclear magnetic resonance of platelet dense granule adenine nucleotides.

31P-NMR spectra of platelets were obtained after depletion of metabolic ATP and ADP by inhibitors. The remaining dense-granule nucleotides show prominent NMR peaks for rabbit, cow and pig, but not human, platelets. Each animal species has a unique and reproducible spectrum reflecting differences in dense-granule compositions, but in all, the dense-granule peaks are broader (by as much as 10-fold) than in spectra of solutions approximating the granule composition. Measurement of the transverse relaxation times, T2, for each resonance band yields intrinsic peak widths which generally are much narrower than observed peak widths. This result indicates that the broad dense-granule peaks are composed of intrinsically narrower peaks of slightly different chemical shifts, implying that the platelet dense granules of a given species are heterogeneous in composition. Our observations suggest that variations in divalent cation ratios and/or pH in the dense granules of a single animal can explain the spread of resonance positions.

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study.

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.

Composition of the aqueous phase of chromaffin granules.

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 +/- 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine:ATP molar ratio (4.41 +/- 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine:protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.

Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.

Isolation of chromaffin cell plasma membranes on polycationic beads.

We have tried to define which proteins of chromaffin cell plasma membranes are facing the cytoplasm by surface labelling a selectively oriented membrane preparation. Viable chromaffin cells were isolated by collagenase treatment of bovine adrenals. Plasma membranes from these cells were isolated on polycationic beads by the method of Jacobson and Branton (Jacobson, B.S. and Branton, D. (1977) Science 195, 302--304). The purity and orientation of the membranes were defined by biochemical and morphological criteria. The membranes, with their external side apposed to the bead surface, were enriched about 10-fold with respect to a whole cell homogenate, and contained only small amounts of contaminating organelles. Surface specific iodination of membranes on beads with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen), followed by polyacrylamide gel electrophoresis, allowed the identification of cytoplasmically exposed proteins. A different pattern was observed when intact cells were labelled prior to membrane isolation. The advantages and possible uses of this immobilized membrane preparation are discussed.

Catecholamine content of chromaffin granule "ghosts' isolated from bovine adrenal glands.

Studies on the mechanism of catecholamine transport into chromaffin granules is complicated by the release of endogenous catecholamines. To overcome this problem chromaffin granule ghosts have been prepared by many investigators by osmotic lysis of the granules which results in a loss of over 90% of the endogenous catecholamine. However, in the studies reported here, the resulting ghosts still contained 36 +/- 3.9 nmol epinephrine/mg of protein if they were lysed by passage through a Sephadex G-50 column preequilibrated with hypoosmotic media. This residual catecholamine was found to slowly diffuse out of the ghosts in a temperature-dependent process at a rate sufficient to interfere with kinetic analysis of catecholamine transport. Attempts to remove the endogenous catecholamine from the ghosts indicated that most of it could not be removed by further osmotic shock of freeze-thaw treatments, but that over 85% of it was released from the granules by incubating them at 30 degree C for 90 min or by dialysis with a 35 and 86% loss of rate of catecholamine transport into the ghosts, respectively. If the endogenous catecholamine was removed from chromaffin granule ghosts by preincubating them for 90 min at 30 degree C, the resulting ghosts transported catecholamine with a linear Lineweaver-Burk plot indicating a Km of 12 +/- 2 microM. In addition, the resulting ghosts did not leak catecholamines over a 10 min period at 30 degree C, and the transport of catecholamines was blocked by reserpine and enhanced with increasing pH from 6.0 to 8.5.

Similarities of physico-chimical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules.

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.

An immunoreactive 8-azido ATP-labeled protein common to the lysosomal and chromaffin granule membrane.

The H+-ATPases of eukaryotic cell organelles, including the rat liver lysosomal (tritosomal) H+-ATPase and the bovine chromaffin granule ATPase, exhibit similarities in function, substrate requirements, and inhibitor responses. We have explored the possibility that these pumps also exhibit immunological similarities, and that common determinants may be present on polypeptides important to function, such as ATP binding. Toward this end, antibodies were produced in rabbits against a highly purified, detergent-solubilized and fractionated chromaffin granule proton pump preparation. This antibody reacted with a 70-80 kDa protein of the lysosomal membrane on Western blots. We have previously shown that photolysis with 8-azido-ATP inhibits lysosomal N-ethylmaleimide-sensitive, vanadate-, ouabain- and oligomycin-insensitive ATP hydrolysis and H+ transport, with concomitant labeling of a 70-80 kDa membrane protein, amongst others. Here, we report that the photolysis with 8-azido-ATP also leads to inhibition of chromaffin granule H+ pump function and pump-related ATP hydrolysis, with concomitant N-ethylmaleimide-sensitive, ATP-protectable, 8-azido-[alpha-32P]ATP labeling. The anti-chromaffin granule antibody reacts with an approx. 70 kDa protein of the chromaffin granule and the lysosome. This raises the possibility that the 70 kDa 8-azido-ATP-reactive, immunologically similar proteins may play a similar role in pump function such as ATP binding and/or hydrolysis in these organelles.

Immunolocalization of betagranin: a chromogranin A-related protein of the pancreatic B-cell.

The tissue and subcellular distribution of betagranin, a chromogranin A-related, cosecreted protein produced in rat insulinoma tissue, has been investigated using a combination of density gradient centrifugation, immunoblotting, immunofluorescence, and immunoelectron microscopic techniques. Antibodies raised to insulinoma betagranin recognized antigens of the same molecular size (approximately 20,000 daltons) in insulinoma tissue and normal islets. Antigenicity was confined principally to secretory granules, and in insulinoma tissue was colocalized with insulin. Within the islet, all endocrine cells were immunoreactive, although subpopulations of beta- and alpha-cells displayed a more intense immunofluorescence. Adrenal tissue and anterior and posterior pituitaries were also highly immunoreactive, the antigen again being confined principally to the secretory granule. Higher molecular size species of 65,000, 85,000, and 100,000 daltons, which predominated in adrenal, were also present in pituitary along with equivalent amounts of the 20,000-dalton proteins. Isolated cells in the gastric antrum, small intestine, and colon were strongly immunofluorescent, but again, the molecular form differed from those of other tissues. Parallel experiments performed with antichromogranin A antisera suggested that betagranin in pancreatic B-cells is formed from chromogranin A by limited proteolysis within the secretory granule. It would appear that although chromogranin A is confined to tissues of the diffuse neuroendocrine system it can be processed differentially in tissues in this series. Potentially, the biological activity of chromogranin A resides in such derived peptides rather than in the parent molecule.

Human chromogranin A. Purification and characterization from catecholamine storage vesicles of human pheochromocytoma.

Chromogranin A is the quantitatively major soluble protein in catecholamine storage vesicles of the adrenal medulla and sympathetic nerve, and has been a useful index of exocytosis during sympathoadrenal neurosecretion. To probe human catecholamine storage and release, we isolated chromogranin A from chromaffin tissue in human pheochromocytoma, and compared it to chromogranin A isolated from chromaffin tissue in bovine adrenal medulla. The preparation included catecholamine storage vesicle isolation by sucrose gradient centrifugation, removal of dopamine-beta-hydroxylase by affinity chromatography on Concanavalin A-Sepharose, and preparative polyacrylamide gel electrophoresis. Human and bovine chromogranin A displayed considerable interspecies homology. Human chromogranin A is a 68,000 dalton monomeric protein with an unusual amino acid composition (31.53 weight % glutamic acid); an acidic, microheterogeneous isoelectric point (4.57-4.68); a characteristic tryptic digest peptide map; and marked dissimilarity to dopamine-beta-hydroxylase in all properties studied. A new probe of human sympathoadrenal function is available in chromogranin A.

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles.

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.

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 be important for targeting of precursors to secretory granules or correct processing.

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38.

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule.

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.

A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore.

Upon SDS PAGE of isolated mediatophore, an acetylcholine-translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue-long peptide completely homologous with a sequence of the proton-translocating proteolipid from bovine chromaffin granules. A 51-mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in lambda Zap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells.

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.

Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide.

Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a beta-galactosidase-preproenkephalin A(69-207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33-5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23.3, 18.2 and 12.6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18.2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.