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1.
The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.  相似文献   
2.
Endogenous protein phosphorylation patterns were compared in occluded and non-occluded cytosol fractions prepared from rat forebrain. The occluded fraction was taken as representative of synaptosomal cytosol. One- and two-dimensional autoradiographs revealed the presence in non-occluded cytosol of a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinase activities of Mr 300kD, corresponding to phosphorylated microtubule-associated protein-2 (MAP-2); this protein was absent in occluded cytosol. In contrast, a major substrate for protein kinase C was observed exclusively in occluded cytosol after phosphorylation under basal conditions. However, after phosphorylation in the presence of exogenous lipids, approximately equal amounts of the 82kD substrate were detected in both fractions, suggesting that protein kinase C in the occluded fraction was present in a partially activated state. Other minor differences in phosphorylation patterns between the two fractions were observed.Special Issue dedicated to Prof. Eduardo De Robertis.  相似文献   
3.
1. The turnover of protein-bound phosphorylserine in preparations of membrane fragments from ox brain cortex was studied. 2. Turnover was considered to arise from the action of intrinsic protein kinases and phosphatases on a membrane protein or proteins. 3. Properties of the kinase system were studied by measuring the rate of incorporation of (32)P from gamma-labelled ATP into protein-bound phosphorylserine isolated from partial acid hydrolysates of membrane proteins. 4. Properties of the phosphatase system were studied by observing the rate of loss of (32)P from membrane preparations pre-labelled with [(32)P]ATP. 5. Net phosphorylation and dephosphorylation of membrane protein was observed during incubation of membrane preparations with and without ATP. 6. The rate of turnover was about 4nmol of P/h per mg of protein at 20 degrees C; dephosphorylation was considered to be the rate-limiting step.  相似文献   
4.
1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from (32)P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ;phosphorylserine' fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [(32)P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ;phosphorylserine' fraction by 53+/-11%, as compared with only 19+/-5% for the phosphorylserine isolated by electrophoresis.  相似文献   
5.
Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li+ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [32P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more 32P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.  相似文献   
6.
Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.  相似文献   
7.
1. Exposure of slices of cerebral cortex from guinea pigs to electrical pulses for 10s or to noradrenaline, 5-hydroxytryptamine or histamine increases the rate of phosphorylation of unidentified proteins in the tissue; the increases in protein phosphorylation due to electrical pulses and noradrenaline were non-additive, whereas the increases due to pulses and 5-hydroxytryptamine or histamine were additive. 2. The stimulating effects of electrical pulses and noradrenaline on protein phosphorylation were antagonized by the beta-adrenergic blocking agents L-propranolol, dichloroisoprenaline, practolol and ICI 66082, but not by the alpha-adrenergic blocking agents, phentolamine and phenoxybenzamine. 3. The increase in protein phosphorylation associated with electrical pulses was antagonized by 10 mum-trifluoperazine and 0.5 mum-prostaglandin E1. 4. It is postulated that under the experimental conditions used the action of electrical pulses on protein phosphorylation is mediated by noradrenaline acting through a beta-adrenergic receptor mechanism probably involving adenylate cyclase.  相似文献   
8.
9.
The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+.  相似文献   
10.
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