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Notes: Abstract— The effects of several inhibitors, including vinblastine and colchicine, on the accumulation of a number of putative transmitters by a rat brain synaptosomal preparation and their subsequent release by excess K+ was examined. In addition, the effect of the alkaloids on the ATPase activity of the actomyosin-like protein, neurostenin, isolated from the synaptosomal preparation, was studied. The uptakes of radioactive glutamate, GABA, dopamine and norepinephrine were energy-dependent, as evidenced by their susceptibility to 0.01 mM carbonyl cyanide m-chlorophenylhydrazone (Cl-CCP), 01 mM ouabain and temperature. The active accumulations of GABA, dopamine and norepinephrine were also greatly inhibited by 1 mM6-hydroxydopamine (6-OHDA), 01 mM mersalyl, 0.05–0.25mM vinblastine and 0.1–1.0 mM colchicine. Vinblastine was approximately 10-fold more potent (K1, −0.1 mM) than colchicine as an inhibitor. The release of actively accumulated dopamine or norepinephrine by excess K+ (increasing the [K+] from 5 to 30 mM) was inhibited somewhat when vinblastine was present during the entire incubation period. If the synaptosomes were preloaded with the radioactive compounds prior to addition of vinblastine, there was no discernible effect on the relative amount of material released by excess K+. However, the addition of inhibitor under the latter conditions caused a leakage of radioactivity into the medium even without excess K+ being present. Glutamate accumulation was somewhat different from that of GABA, dopamine or norepinephrine. Although it required energy for uptake, 6-OHDA, mersalyl, vinblastine or colchicine were not inhibitory. Studies of the oxidative metabolism of glutamate and GABA by this synaptosomal preparation indicated that the mechanisms of inhibition by vinblastine was not attributable to a metabolic effect. Both vinblastine and colchicine inhibited the Mg2+-stimulated, but not the Ca2+-activated ATPase of neurostenin. This effect was probably attributable to an interaction of the vinblastine with the neurin moiety of this actomyosin-like protein. We suggest that the inhibitory phenomena exhibited by vinblastine and colchicine in this synaptosomal preparation arose from the effect of these alkaloids on the neurin associated with the synaptic membrane.

Notes: Abstract— An actomyosin-like protein (neurostenin) has been isolated from the synaptosomal fraction of bovine and rat brains. A similar protein could not be obtained from the mitochondrial, microsomal, myelin or supernatant fractions. The synaptosomal protein is a Mg2+ -Ca2+ -stimulated ATPase which exhibited the phenomenon of superprecipitation and viscosimetric sensitivity to ATP characteristic of actomyosins. It constituted 8–10 per cent of the total synaptosomal proteins. The protein could be dissociated into actin-like (neurin) and myosin-like (stenin) proteins by ultracentrifugation in sucrose gradients containing KI and ATP. The neurin, as well as muscle actin, stimulated the Mg2+ -ATPase activity of stenin and muscle myosin; the relative viscosities of the mixtures were increased and became sensitive to added ATP. The neurin contained 0.9 mol of 3-methylhistidine per 50,000 g of protein. The presence of these proteins in the synaptosomes suggests the possibility that they participate in nerve-ending functions, e.g. release of transmitter materials.

Notes: Abstract— Paper chromatography of extracts from mesenteric Pacinian corpuscles of the cat revealed the presence of glutamic acid, glutamine, aspartic acid and alanine as major amino acids, and glycine, serine and threonine in traces; GABA was not detected. Levels of glutamic acid (0·75 μmol/g ′ 0·37, s.d.), glutamine (1·34 ± 0·55), and aspartic acid (0·32 ± 0·22) of mesenteric and pancreatic samples of Pacinian corpuscles were determined by separation on chromatographic columns. The protein values averaged 5·2 ± 0·66 per cant of the wet weight.Treatment of the cats with reserpine or pargyline or deafferentation of the Pacinian corpuscles did not significantly alter these values.

Notes: Abstract— —In the head of the caudate nucleus, the relative specific activity of glutamine (glutamic acid specific activity = 1) was less than 1 with intravenous [14C]leucine as the tracer metabolite. This is in contrast to observations made in other brain areas (cortex, hippocampus, thalamus, pons, and medulla) where the relative specific activity of glutamine was greater than 1. This is also in contrast to findings when [l-14C]acetate was utilized as the tracer; under these conditions, in all brain areas, including the head of the caudate nucleus, the relative specific activity of glutamine was greater than 1. It is inferred that the differences in metabolism of [14C]leucine and [14C]acetate in the head of the caudate from that in other brain areas reflect differences in compartmentation of the glutamate-glutamine system.

Notes: The association of γ-aminobutyric acid (GABA) with rat brain synaptic membranes is complex and involves more than one set of binding sites. This fact is suggested by the form of experimentally determined binding curves and confirmed by the destruction of one set of homogenous sites by covalent modifications with iodine. The difference in binding of GABA by two sets of membranes which have been exhaustively iodinated in the presence and absence of an excess of GABA corresponds to 1/6 of the binding expressed by uniodinated membranes after equilibration with 0.25 μm-GABA. This difference appears to be the result of the expression of a homogeneous set of independent sites, each of which contains an iodinatable'residue that is protected by bound GABA.Examination of the factors leading to the retention of radioactive ligand by a synaptic membrane preparation is suggestive of a method for determining the maximum number of binding sites for a given ligand and for evaluating solution space without displacing the bound species or adding a presumably inert tracer. Three additional measurements of retention of high ligand concentration are shown to be sufficient for the determination. Employment of the method facilitates the investigation of the interaction of the membrane preparations with individual agonists and antagonists.

Notes: Affinity chromatography was used to partially purify the troponin complex from crude regulatory proteins obtained from bovine brain cortex. Three components were obtained from this partially purified troponin complex by treatment with 6 M-urea and 1 mM-EGTA followed by chromatography on DEAE-Sephadex-A50. The effects of the three components on skeletal muscle actin activated MgATPase activity of muscle myosin (ATP phosphohydrolase, EC 3.6.1.3.) suggested that they were analogous to that of the skeletal muscle troponins I, C, and T. The apparent molecular weights of the brain troponin subunits (I, C, and T) were 18, 700, 14, 000 and 36, 400, respectively. The molecular weights of the former two proteins were less than those reported for the analogous skeletal muscle troponins. Thus, brain actomyosin complex may be regulated in a manner similar to that of striated muscle actomyosin.