A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide‐containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal‐containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well‐characterized membrane protein of small vesicles in neurons and endocrine cells.

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Communicated by Alfred G. Gilman, December 22, 1989 (received for review December 6, 1989) ABSTRACT rab3, a low molecular weight GTP-binding protein, is primarily expressed in brain, where it is present in soluble and membrane-bound forms. Membrane-bound rab3 in brain is exclusively localized on synaptic vesicles, the secretory organelles of the synapse that store and release neurotransmitters. rab3 is also expressed in endocrine tissues such as the adrenal medulla, where it is found together with other synaptic vesicle proteins on microvesicles distinct from chromaffin granules. The tight binding of rab3 to membranes correlates with hydrophobic modifications that are different in the membrane-bound and soluble forms of rab3. The results demonstrate the exclusive targeting of a small GTP-binding protein to secretory vesicles of a subset of the regulated pathway of secretion.

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The push-pull cannula technique was used to evaluate the role of the medial preoptic/anterior hypothalamic area (MPO) in regulating pituitary luteinizing hormone (LH) and prolactin release. The concentrations of the three catecholamines – dopamine, norepinephrine (NE), epinephrine (E) – and gamma-aminobutyric acid (GABA) and glutamate could be measured in 15-min fractions at which interval blood samples for LH and prolactin determination were also collected. Comparison of neurotransmitter release rates into the MPO were made between ovariectomized and ovariectomized estradiol benzoate treated rats. Release of the neurotransmitters occurred in a pulsatile manner, the release episodes for each transmitter appeared to be independent of the others. No direct correlation between neurotransmitter release episodes and blood LH or prolactin levels could be established. The release of GABA was significantly lower and that of NE and E higher in ovariectomized animals in comparison to estrogen-primed ovariectomized animals under negative feedback conditions. In the afternoon, however, when the estrogen stimulated LH and prolactin release, preoptic GABA release was low, whereas preoptic NE and particularly E release rates were high. Conspicuously high dopamine and NE release episodes were observed in estrogen-primed animals at noon, i.e., prior to the expression of the positive feedback signal. This may reflect a biochemical correlate to the so-called critical period. No consistent differences between ovariectomized and ovariectomized estradiol-17β benzoate treated animals were observed for preoptic glutamate release rates. The data show that preoptic GABA release rates show generally an inverse pattern to NE and E release and therefore also to blood LH and prolactin levels. No direct mathematical correlation between any of the neurotransmitter release rates and blood hormone levels could be established.

Abstract. A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 ceils with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins.P29 has an isoelectric point of ,',,5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.