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1.
KS-504a inhibited bovine brain calmodulin-dependent cyclic nucleotide phosphodieaterase (CaM-PDE) with an IC 50 value of 122 μ m. The inhibition was reversed by a high concentration of calmodulin. Cal modulin-independent activities of the enzyme were not affected by the compound at the same concentration ranges. Ca 2+-dependent interaction of the compounds with calmodulin was shown using hydrophobic fluorescence probes. These data indicated that the compound exerted its effects on CaM-PDE by interacting with calmodulin. KS-504a also inhibited other calmodulin-dependent enzymes at different concentration ranges; myosin light chain kinase was inhibited at the lowest concentrations with an IC 50 value of 6.3 μ m. The inhibition mechanism was competetive with respect to calmodulin and non-competetive to ATP. 相似文献
2.
Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent M r of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca 2+ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase. 相似文献
3.
The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish ( Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca 2+ concentration. Stimulation was marked at low, nonsaturating Ca 2+ concentrations and decreased increasing Ca 2+, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca 2+. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca 2+, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents. 相似文献
4.
The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca 2+ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca 2+ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca 2+ and cAMP signalling pathways. 相似文献
5.
Cd 2+ provokes inositol trisphosphateproduction and releases stored Ca 2+, apparently by binding to a zinc site in the external domain of an orphan receptor. One pM Cd 2+ evokes an immediate spike in cytosolic free Ca 2+, which is similar to that evoked by bradykinin. Platelet-derived growth factor (PDGF) also increases free Ca 2+ in human dermalfibroblasts, but there is a distinct lag before free Ca 2+ rises in response to PDGF. Genistein, which selectively inhibits tyrosine kinases, markedly inhibited Ca 2+ mobilization evoked by PDGF. Calcium mobilization triggered by cadmium or bradykinin was relatively insensitive to genistein. The PDGF receptor is known to be a tyrosine kinase, whichphosphorylates and thereby activatesphospholipase C, whereas a G protein couples the bradykinin receptor to anotherphospholipase C isoform. These findings support the hypothesis that the orphan receptor triggered by cadmium is coupled to phospholipase C via a G protein.Abbreviations BSA
bovine serum albumin
- BK
bradykinin
- [Ca 2+] i
cytosolic free calcium
- DME
Dulbecco's modified Eagle's medium
- FBS
fetal bovine serum
- HEPES
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid
- IC 50
concentration that produces 50% inhibition
- PDGF
platelet-derived growth factor
- PSS
physiological salts solution
- SE
standard error of the mean 相似文献
6.
Phospholipid-sensitive and calmodulin-sensitive Ca 2+-dependent phosphorylation of a number of endogenous proteins in the soluble and particulate fractions of the rat cerebral cortex was inhibited by phenothiazine antipsychotic drugs. The mean IC 50 values (concentrations causing 50% inhibition of phosphorylation) for trifluoperazine, chlorpromazine and fluphenazine were 16, 24 and 27 μM, respectively. Dibucaine, a local anesthetic drug, was much less effective. It appears that these neuroleptic agents may be useful tools for the study of Ca 2+-dependent protein phosphorylating systems regulated by either phospholipid or calmodulin in the brain. 相似文献
7.
A novel M r 17,000 Ca 2+-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca 2+- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca 2+-binding proteins on protein kinase C activity indicated that the M r 17,000 Ca 2+-binding protein was the most potent inhibitor, followed by the intestinal Ca 2+-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M r 21,000 Ca 2+-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M r 17,000 Ca 2+-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M r 17,000 Ca 2+-binding protein, and possibly other Ca 2+-binding proteins, may play a physiological role in regulating the activity of protein kinase C. 相似文献
8.
A soluble Ca 2+/calmodulin dependent protein kinase has been partially purified (~400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC 50 of 1.5 and 0.25 m respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca 2+/ calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism. 相似文献
9.
The effect of regucalcin on Ca 2+/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl 2 (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca 2+/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca 2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca 2+ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca 2+/calmodulin-dependent protein kinase activity in renal cortex cells. 相似文献
10.
The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C 1 phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C 2 phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca 2+ /phosphatidylserine/diolein than by Ca 2+ alone or Cal 2+ /calmodulin (group C 1) or was completely dependent upon Ca 2+ /phosphatdylserine/diolein (group C 2); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca 2+; (d) NaCl, which inhibited cAMP- and Ca 2+/calmodulindependent phosphorylation, slightly increased phosphorylation of group C 1 and slightly decreased phosphorylation of group C 2 phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20–30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C 2 phosphoproteins were considerably more sensitive. The IC 50 for inhibition of P96 labeling was 19 μM. but for P83 was 190 μM. Group B phosphoproteins were also slightly inhibited, and the IC 50 for P63 was 290 μM. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [ 3H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C. 相似文献
11.
A calcium-dependent protein kinase activity from suspension-cultured soybean cells ( Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45Ca 2+ in the presence of KCl and MgCl 2, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity. 相似文献
12.
Ca 2+-calmodulin tubulin kinase activity was isolated from brain cytosol and separated from its substrate protein, tubulin, and Ca 2+ regulatory protein, calmodulin. Characterization of the Ca 2+-tubulin kinase system revealed a Km of 4 μM, 0.5 μM, 60 μM for Ca 2+, calmodulin and ATP, respectively. The tubulin kinase system bound to a calmodulin affinity column in the presence of Ca 2+ and was released from the column by chelation with EGTA. A major 55,000 and a minor 65,000 dalton peptide were identified as the only calmodulin binding proteins in the enzyme fraction, indicating that one or both of these peptides represent the calmodulin binding subunit of the Ca 2+-calmodulin tubulin kinase system. 相似文献
13.
In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated Mr of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca 2+ and calmodulin. Peak II revealed a basal activity in the absence of Ca 2+ and calmodulin, which was stimulated 2-fold by addition of Ca 2+. Calmodulin had no effect on the peak II activity. 相似文献
14.
A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca 2+-dependent cyclic AMP phosphodiesterase of . The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca 2+,Mg 2+)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca 2+-dependent fashion with an apparent K a of 5 × 10 ?5. The factor and brain calmodulin had no effect on the phosphodiesterase of . It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes. 相似文献
15.
Evidence for the involvement of Ca 2+ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U- 14C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U- 14C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca 2+, whereas lipolytic acyl hydrolase proved to be insensitive to Ca 2+. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca 2+. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC 50 values ranging from 10 to 15 micromolar. Thus the Ca 2+-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca 2+ on phospholipase D is independent of calmodulin. The role of Ca 2+ as a second messenger in the initiation of membrane lipid degradation is discussed. 相似文献
16.
Ca 2+ plays a major role in neurotransmission and synaptic modulation. Evidence is presented to support the calmodulin hypothesis of neurotransmission developed in this laboratory stating that calmodulin, a major Ca 2+ binding protein in brain, mediates the effects of Ca 2+ on neurotransmission. Calmodulin was isolated from highly enriched preparations of synaptic vesicles and nerve terminal cytoplasm. Ca 2+ and calmodulin were shown to regulate several synaptic processes in isolated and intact preparations, including endogenous synaptic Ca 2+-calmodulin protein kinase activity, neurotransmitter release, and synaptic vesicle and synaptic membrane interactions. Ca 2+ and calmodulin were shown to activate a synaptic tubulin kinase system which was shown to be a distinct enzyme system from the cyclic AMP protein kinase. Ca 2+ and calmodulin stimulated phosphorylation of tubulin altered the properties of tubulin, forming insoluble tubulin fibrils. Evidence for the role of Ca 2+-calmodulin kinase activity, especially the calmodulin-tubulin kinase, in neurotransmission are presented. The effects of several neuroactive drugs on the synaptic calmodulin system are presented. The results support the hypothesis that calmodulin mediates many of calcium's actions at the synapse, and that the effects of Ca 2+ on synaptic protein phosphorylation, especially synaptic tubulin, may provide a biochemical mechanism for converting the Ca 2+ signal into a motor force in the process of neurotransmission. 相似文献
17.
Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca 2+-dependent protein kinase from pig testis. Gossypol inhibited Ca 2+-dependent activity of the enzyme without affecting its basal activity. The IC 50 value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP ( Ki = 31 μM) or lysine-rich histone ( Ki = 30 μM), and competitively with respect to phosphatidylserine ( Ki = 2.1 μM). With Ca 2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca 2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC 50 value of 88 μM. These results suggested that a phospholipid-sensitive Ca 2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis. 相似文献
18.
A multifunctional Ca 2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca 2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca 2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca 2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca 2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca 2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca 2+/calmodulin-dependent protein kinase of Candida albicans. 相似文献
19.
The characteristics of Ca 2+ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca 2+ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0. The Ca2+-dependent modulation protein, calmodulin, was tested for its effect on Ca2+ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca2+ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca2+ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca2+ transport activity. The inhibition of Ca2+ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca2+ uptake into root endoplasmic reticulum. 相似文献
20.
A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540–3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of cadmodulin did not require Ca 2+ for activation of the enzyme; and activation occured in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca 2+. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased Vmax and with no changes in the Fm (ATP). High levels of cation, up to 100 mM Mg 2+, did not effect the action of cadmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue. 相似文献
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