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1.
We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+. 相似文献
2.
Phosphorylation of Synaptic Membrane Glycoproteins: The Effects of Ca2+ and Calmodulin 总被引:1,自引:0,他引:1
Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four-fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase. 相似文献
3.
Abstract: Purified rat brain Ca2+ /calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+ /calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+ -independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC50 =0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+ /CaM on native CaM-kinase II and on peptide CaMK 281-309. 相似文献
4.
Tubulin-Associated Calmodulin-Dependent Kinase: Evidence for an Endogenous Complex of Tubulin with a Calcium-Calmodulin-Dependent Kinase 总被引:6,自引:4,他引:2
James R. Goldenring James E. Casanova Robert J. DeLorenzo 《Journal of neurochemistry》1984,43(6):1669-1679
A Ca2+ -calmodulin kinase that phosphorylates tubulin and microtubule-associated proteins as major substrates has been purified and characterized from brain cytoplasm. It is important to determine if cytoskeletal proteins are major natural substrates for this kinase system. This report demonstrates that a significant fraction of brain cytosolic calmodulin-dependent kinase activity exists in tight association with tubulin in the form of a stable complex. The tubulin-calmodulin kinase complex displayed an apparent molecular weight on gel filtration of approximately 1.8 X 10(6) daltons. The specific activity of tubulin kinase in the complex was enriched over 20-fold in comparison with brain cytosol. Although purified tubulin alone did not adhere to a calmodulin column, the tubulin associated with the calmodulin kinase complex did bind specifically to the calmodulin affinity resin. The kinase activity was shown to be tightly associated in complex with tubulin by (1) copurification, (2) isolation on gel filtration chromatography, (3) isolation on ion-exchange chromatography, and (4) binding to calmodulin. The kinase complexed with tubulin was identical to the previously purified kinase as judged by several criteria including (1) subunit molecular weights, (2) isoelectric points, (3) autophosphorylation characteristics, (4) calmodulin binding properties, (5) kinetic parameters of tubulin phosphorylation, (6) phosphoamino acid phosphorylation sites on alpha- and beta-tubulin, and (7) identical subunit 125I-tryptic peptide maps. The results indicate that a significant fraction of this previously purified calmodulin kinase is endogenously associated with tubulin in brain cytoplasm and may play a role in mediating some of the effects of calcium on neuronal function. 相似文献
5.
Hideyuki Yamamoto Nobuaki Maeda Michio Niinobe Eishichi Miyamoto Katsuhiko Mikoshiba† 《Journal of neurochemistry》1989,53(3):917-923
Purified P400 protein was phosphorylated by both purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Because P400 protein was suggested to function as an integral membrane protein, we investigated the phosphorylation of P400 protein using crude mitochondrial and microsomal fractions (P2/P3 fraction). Incubation of the P2/P3 fraction from mouse cerebellum with cyclic AMP or the catalytic subunit of A-kinase stimulated the phosphorylation of P400 protein. The phosphorylation of P400 protein was not observed in the P2/P3 fraction from mouse forebrain. Cyclic AMP and A-kinase enhanced the phosphorylation of several proteins, including P400 protein, suggesting that P400 protein is one of the best substrates for A-kinase in the P2/P3 fraction. Although endogenous and exogenous CaM kinase II stimulated the phosphorylation of some proteins in the P2/P3 fraction, the phosphorylation of P400 protein was weak. Immunoprecipitation with the monoclonal antibody to P400 protein confirmed that the P400 protein itself was definitely phosphorylated by the catalytic subunit of A-kinase and CaM kinase II. A-kinase phosphorylated only the seryl residue in P400 protein. Immunoblot analysis of the cells in primary culture of mouse cerebellum confirmed the expression of P400 protein, which migrated at the same position on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that in the P2/P3 fraction. Incubation of the cultured cerebellar cells with [32P]orthophosphate resulted in the labeling of P400 protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Stefan Strack Mary Ann Barban Brian E. Wadzinski Roger J. Colbran 《Journal of neurochemistry》1997,68(5):2119-2128
Abstract: Autophosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) at Thr286 generates Ca2+ -independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr286 -autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286 -autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32 P autophosphorylated in the presence of Ca2+ /calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2+ -independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments. 相似文献
7.
Regulation of the Actin-Activated Mg-ATPase of Brain Myosin via Phosphorylation by the Brain Ca2+ , Calmodulin-Dependent Protein Kinases 总被引:2,自引:0,他引:2
Etsuro Tanaka Kohji Fukunaga Hideyuki Yamamoto Takafumi Iwasa Eishichi Miyamoto 《Journal of neurochemistry》1986,47(1):254-262
We have previously isolated two Ca2+, calmodulin-dependent protein kinases with molecular weights of 120,000 (120K enzyme) and 640,000 (640K enzyme), respectively, by gel filtration analysis from rat brain. Chicken gizzard myosin light-chain kinase and the 120K enzyme phosphorylated two light chains of brain myosin, whereas the 640K enzyme phosphorylated both the two light chains and the heavy chain. The phosphopeptides of the light chains digested by Staphylococcus aureus V8 protease were similar among chicken gizzard myosin light-chain kinase, the 120K enzyme, and the 640K enzyme. Only the seryl residue in the light chains and the heavy chain was phosphorylated by the enzymes. The phosphorylation of brain myosin by any of these enzymes led to an increase in actin-activated Mg-ATPase activity. The results suggest that brain myosin is regulated by brain Ca2+, calmodulin-dependent protein kinases in a similar but distinct mechanism in comparison with that of smooth muscle myosin. 相似文献
8.
Effect of Monovalent Cations on Na+ /Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes 总被引:1,自引:0,他引:1
Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations. 相似文献
9.
R. Scott Turner C.-H. Jen Chou† Robert F. Kibler† J. F. Kuo 《Journal of neurochemistry》1982,39(5):1397-1404
Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+ , and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca2+ -dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca2+ -dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca2+ -dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7). 相似文献
10.
Phosphorylation of the Postsynaptic Density Glycoprotein gp 180 by Ca2 +/Calmodulin-Dependent Protein Kinase 总被引:2,自引:0,他引:2
James W. Gurd 《Journal of neurochemistry》1985,45(4):1128-1135
Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180. 相似文献
11.
Kinetic Characterization of Ca2+ Transport in Synaptic Membranes 总被引:2,自引:0,他引:2
Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane. 相似文献
12.
Calmodulin and Ca2+ - and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland 总被引:1,自引:0,他引:1
Yasuyuki Hatada Masahide Munemura Kohji Fukunaga Hideyuki Yamamoto Masao Maeyama Eishichi Miyamoto 《Journal of neurochemistry》1983,40(4):1082-1089
Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration. 相似文献
13.
The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed. 相似文献
14.
Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+ /Calmodulin- and cAMP-Dependent Protein Kinases 总被引:2,自引:0,他引:2
Nathan Moskowitz Adam Glassman Christine Ores William Schook Saul Puszkin 《Journal of neurochemistry》1983,40(3):711-718
Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals. 相似文献
15.
Independent protein kinases in the synaptic junction (SJ) isolated from rat cerebrum were characterized. SJ showed a protein kinase activity, phosphorylating intrinsic proteins, even in the absence of cyclic AMP or Ca2+ plus calmodulin (CaM) exogenously added. The activity was affected neither by Ca2+ concentrations in the physiological fluctuation range nor by the addition of specific ligands such as glutamate, aspartate, acetylcholine, and concanavalin A. The activity was not due to cyclic AMP-dependent protein kinase in SJ, since the activity was not inhibited by an inhibitor protein for cyclic AMP-dependent protein kinase, and since synapsin I was not specifically phosphorylated whereas cyclic AMP-dependent kinase appeared to phosphorylate selectively the protein in SJ. Phosphorylation of SJ proteins by the independent kinases was about one-third of that of the Ca2+/CaM-dependent protein kinase intrinsic to SJ. The apparent Km for ATP was estimated to be 700 microM. Proteins of 16K Mr and 117K Mr were specifically phosphorylated under the basic condition (in the absence of the substances known to activate specifically protein kinases), as well as six other proteins both under the basic conditions and in the presence of Ca2+ and CaM. The phosphorylation of 150K Mr, 60K Mr, 51K Mr, and 16K Mr SJ proteins was enhanced after prephosphorylation of SJ proteins by intrinsic kinase in the presence of Ca2+ and CaM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Paul T. Kelly Rick K. Yip Steven M. Shields Michael Hay 《Journal of neurochemistry》1985,45(5):1620-1634
Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication. 相似文献
17.
18.
Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes 总被引:2,自引:0,他引:2
The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization. 相似文献
19.
Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin-deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2+ + Mg2+ -ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin. 相似文献
20.
Mireille Ehret Christopher D. Cash Michel Hamon Michel Maitre 《Journal of neurochemistry》1989,52(6):1886-1891
Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [gamma-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50 microM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation. 相似文献