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1.
We have previously shown that pea seedling plasma membrane contains a protein kinase whose activity towards plasma membrane proteins is markedly increased by micromolar concentrations of calcium and calmodulin. The evidence in this paper suggests that this protein kinase autophosphorylates, that autophosphorylation is increased in a calcium and calmodulin dependent fashion and that prior autophosphorylation modifies activity towards an exogenous substrate. This protein kinase has a molecular weight of approximately 18,000 and is referred to as phosphoprotein18 (pp18). 相似文献
2.
Once ingested by mosquitoes, malaria parasites undergo complex cellular changes. These include zygote formation, transformation of zygote to ookinete, and differentiation from ookinete to oocyst. Within the oocyst, the parasite multiplies into numerous sporozoites. Modulators of intracellular calcium homeostasis, MAPTAM, and TMB-8 blocked ookinete development as did the calmodulin (CaM) antagonists W-7 and calmidazolium. Ca(2+)/CaM-dependent protein kinase inhibitor KN-93 also blocked zygote elongation, while its ineffective analog KN-92 did not have such effect. In vitro both zygote and ookinete extracts efficiently phosphorylated autocamtide-2, a classic CaM kinase substrate, which could be blocked by calmodulin antagonists W-7 and calmidazolium and CaM kinase inhibitor KN-93. These results demonstrated the presence of calmodulin-dependent CaM kinase activity in the parasite. KN-93-treated parasites, however, expressed the ookinete-specific enzyme chitinase and the ookinete surface antigen Pgs28 normally, suggesting that the morphologically untransformed parasites are biochemically mature ookinetes. In mosquitoes, KN-93-treated parasites did not develop as oocysts, while KN-92-treated parasites produced similar numbers of oocysts as controls. These data suggested that in Plasmodium gallinaceum morphological development of zygote to ookinete, but not its biochemical maturation, relies on Ca(2+)/CaM-dependent protein kinase activity and demonstrated that the morphological differentiation is essential for the further development of the parasite in infected blood-fed mosquitoes. 相似文献
3.
T C Chan 《Journal of cellular physiology》1989,141(3):461-466
Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy. 相似文献
4.
Dephosphorylation of neuromodulin by calcineurin 总被引:8,自引:0,他引:8
Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons. 相似文献
5.
J C Abougou M Hagiwara T Hachiya M Terasawa H Hidaka D J Hartshorne 《FEBS letters》1989,257(2):408-410
The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulin-dependent protein kinase II. 相似文献
6.
M Nakane J Mitchell U F?rstermann F Murad 《Biochemical and biophysical research communications》1991,180(3):1396-1402
Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation. 相似文献
7.
O Holian C T Bombeck L M Nyhus 《Biochemical and biophysical research communications》1980,95(2):553-561
The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system. 相似文献
8.
Pierre N. E. De Graan Albrecht Moritz Marina de Wit Willem Hendrik Gispen 《Neurochemical research》1993,18(8):875-881
Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain. 相似文献
9.
The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2. 相似文献
10.
The cardiac sarcolemmal 15-kDa protein, previously shown to be the principal sarcolemmal substrate phosphorylated in intact heart in response to beta-adrenergic stimulation (Presti, C. F., Jones, L. R., and Lindemann J. P. (1985) J. Biol. Chem. 260, 3860-3867), was demonstrated to be the major substrate phosphorylated in purified canine cardiac sarcolemmal vesicles by an intrinsic protein kinase C activity. The intrinsic protein kinase C, detected by its ability to phosphorylate H1 histones, was most concentrated in cardiac sarcolemmal vesicles and absent from sarcoplasmic reticulum membranes. Unmasking techniques localized the intrinsic protein kinase activity and its principal endogenous substrate, the 15-kDa protein, to the cytoplasmic surfaces of sarcolemmal vesicles; phospholamban contaminating the sarcolemmal preparation was not significantly phosphorylated. The intrinsic protein kinase C required micromolar Ca2+ for activity, but not calmodulin. Half-maximal phosphorylation of the 15-kDa protein occurred at 10 microM Ca2+; optimal phosphorylation of the 15-kDa protein by protein kinase C and Ca2+ was additive to that produced by cAMP-dependent protein kinase. Exogenous phospholipids were not required to activate endogenous protein kinase C. However, heat-treated sarcolemmal vesicles, in which intrinsic protein kinase activities were inactivated, were sufficient to maximally activate soluble protein kinase C prepared from rat brain, suggesting that all the necessary phospholipid cofactors were already present in sarcolemmal vesicles. Of the many proteins present in sarcolemmal vesicles, only the 15-kDa protein was phosphorylated significantly in heat-inactivated sarcolemmal vesicles by soluble protein kinase C, confirming that the 15-kDa protein was a preferential substrate for this enzyme. Consistent with a protein kinase C activity in sarcolemmal vesicles, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated 15-kDa protein phosphorylation severalfold, producing approximately 70% of the maximal phosphorylation even in the absence of significant ionized Ca2+. The results are compatible with an intrinsic protein kinase C activity in sarcolemmal vesicles whose major substrate is the 15-kDa protein. 相似文献
11.
Summary Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvae kinase indicate it is the M2 isoenzyme and that its phosphorylation does not affect its catalytic activity. 相似文献
12.
In rat adrenal glomerulosa cells, endogenous substrate proteins for Ca2+/calmodulin (CaM)-dependent protein kinase (glomerulosa CaM kinase) and Ca2+/phospholipid-dependent protein kinase (protein kinase C) were investigated. In a 105,000 g-supernatant fraction (cytosol), the Mr 100,000 protein was phosphorylated in the presence of calcium (calculated free Ca2+ concentration, 460 microM) alone or calcium and CaM, and the phosphorylation of this protein was completely inhibited by the CaM antagonists pimozide (500 microM) and melittin (5 microM) in the presence of calcium alone, respectively. These results indicate that the Mr 100,000 protein is a major substrate for glomerulosa CaM kinase, and considerable amounts of endogenous CaM might be present in the cytosol. In the presence of phospholipids (the micelles of 8 micrograms of phosphatidyl serine and 1 microgram of diacylglycerol), at least twelve proteins of Mr 127,000, 80,000, 70,000, 36,000, 35,000, 33,000, 32,000, 30,000, 27,000, 22,000, 19,000 and 17,000 were phosphorylated, and the phosphorylation of these proteins was enhanced by the addition of calcium, indicating that these proteins are substrates for protein kinase C. No endogenous protein phosphorylation was found in a 105,000 g-particulate fraction. Thus, these findings demonstrate that adrenal glomerulosa cells have specific substrate proteins for glomerulosa CaM kinase and protein kinase C, respectively. 相似文献
13.
Intracellular cell signaling cascades of protozoan parasite Plasmodium falciparum are not clearly understood. We have reported previously (Kumar, A., Vaid, A., Syin, C., and Sharma, P. (2004) J. Biol. Chem. 279, 24255-24264) the identification and characterization of a protein kinase B-like enzyme in P. falciparum (PfPKB). PfPKB lacks the phosphoinositide-interacting pleckstrin homology domain present in mammalian protein kinase B. Therefore, the mechanism of PfPKB regulation was expected to be different from that of the host and had remained unknown. We have identified calmodulin (CaM) as the regulator of PfPKB activity. A CaM binding domain was mapped in the N-terminal region of PfPKB. CaM, in a calcium-dependent manner, interacts with this domain and activates PfPKB. CaM associates with PfPKB in the parasite and regulates its activity. Furthermore phospholipase C acts as an upstream regulator of this cascade as it facilitates the release of calcium from intracellular stores. This is one of the first multicomponent signaling pathways to be dissected in the malaria parasite. 相似文献
14.
Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction 总被引:1,自引:0,他引:1
Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function. 相似文献
15.
Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42. 总被引:14,自引:0,他引:14
E R Chapman D Au K A Alexander T A Nicolson D R Storm 《The Journal of biological chemistry》1991,266(1):207-213
Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine. 相似文献
16.
S Pontremoli B Sparatore F Salamino R De Tullio R Pontremoli E Melloni 《Biochemical and biophysical research communications》1988,151(1):590-597
The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state. 相似文献
17.
Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas 总被引:3,自引:0,他引:3
J A Cohn B Kinder J D Jamieson N G Delahunt F S Gorelick 《Biochimica et biophysica acta》1987,928(3):320-331
A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function. 相似文献
18.
Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates 总被引:2,自引:2,他引:0
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Roberts DM 《Plant physiology》1989,91(4):1613-1619
By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin-dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a Vmax/Km ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity. 相似文献
19.
Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban. 相似文献
20.
R Deswal G K Pandey M R Chandok N Yadav A Bhattacharya S K Sopory 《European journal of biochemistry》2000,267(11):3181-3189
A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP. 相似文献