RESUMEN
A practical, gram-scale synthesis of L-pyrazinylalanine (Paa) is described, utilizing Schöllkopf's D-valine-derived bis-lactim ether chiral auxiliary.
Asunto(s)
Alanina/análogos & derivados , Alanina/química , Alanina/síntesis química , Pirazinas/química , Pirazinas/síntesis química , Estructura Molecular , EstereoisomerismoRESUMEN
High molecular mass polypeptides (Mr greater than 100,000) of plain synaptic vesicles from bovine cerebral cortex were separated using porous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Four major bands, of Mr 262,000, 249,000, 216,000, and 173,000, were resolved. Investigations into the membrane association of the Mr 216,000 and 173,000 proteins by means of solubilization experiments and Sepharose 4B chromatography indicate that the former is a peripheral protein and the latter is more firmly attached, possibly an integral protein. Finally, the Mr 216,000 protein was shown to be highly enriched in synaptic vesicles compared to other brain subfractions. It thus appears to be specifically associated with synaptic vesicles and therefore may have an important role specific to synaptic vesicle function or structure.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Bovinos , Clatrina/metabolismo , Electroforesis en Gel de Poliacrilamida , Mitocondrias/metabolismo , Peso Molecular , Espectrina/metabolismo , Membranas Sinápticas/metabolismo , Sinaptosomas/metabolismoRESUMEN
Calf brain plain synaptic vesicle proteins have been cross-linked with bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone, a homobifunctional, cleavable reagent, as well as with N-hydroxysuccinimidyl 4-azidobenzoate, a photosensitive, heterobifunctional reagent. These results demonstrate the generality of a recent report that synaptic vesicle proteins can be cross-linked, in contrast to a prior report that no cross-linking could be observed. The reagents gave some differences in the proteins that were preferentially cross-linked. A protein at Mr = 173 000, which comigrates with clathrin, is present in the plain synaptic vesicle fraction and appears to be involved in cross-linking. A high degree of association and structural organization of synaptic vesicle proteins is suspected, since extensive cross-linking of most synaptic vesicle proteins with high-molecular-mass proteins, which are probably structural in nature, is observed. A protein with an Mr of 249 000 is specifically cross-linked to a protein of Mr 42 000, probably actin, suggesting that the 249 000-Mr protein may be a spectrin-like molecule. The present results suggest that synaptic vesicles may be organized by a spectrin-like matrix similar to that observed in erythrocytes and other cells.
Asunto(s)
Química Encefálica , Proteínas del Tejido Nervioso/análisis , Vesículas Sinápticas/análisis , Animales , Azidas , Bovinos , Reactivos de Enlaces Cruzados , Densitometría , Electroforesis/métodos , Coloración y Etiquetado , SuccinimidasRESUMEN
Concanavalin A was employed as a tool to investigate the organization of synaptic vesicle glycoproteins. The lectin was incubated in the presence of both intact and Triton X-100 treated calf brain synaptic vesicles. Electrophoresis of treated membranes clearly demonstrated that the majority of Concanavalin A binding sites was not exposed in intact synaptic vesicles. The vesicles were isolated by the procedure of DeLorenzo and Freedman (DeLorenzo, R. J., and Freedman, S. D. (1978) Biochem. Biophys. Res. Commun. 80, 183-192). However, an extra centrifugation step at 55,000 g was required to obtain plain vesicles nearly free from coated vesicles and membrane fragments.