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1.
The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent Km of the enzyme for the substrate acetyl-CoA without showing any significant effect on the Vmax of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.  相似文献   

2.
Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, Vmax of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.  相似文献   

3.
Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A2 inhibitors 4-bromophenacyl bromide and glucocorticosteroids prednisolone and dexamethasone attenuate Ca2+ responses induced by neuroleptic trifluoperazine in macrophages. The results suggest the involvement of phospholipase A2 and arachidonic acid metabolism cascade in the effect of trifluoperazine on intracellular Ca2+ concentration in macrophages.  相似文献   

4.
K.S. Cheah  J.C. Waring 《BBA》1983,723(1):45-51
The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca2+-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the ADPO and Ca2+O ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca2+-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c1 and cytochrome c1-aa3 segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c1 by 92% with succinate as substrate, and of cytochrome c and cytochrome aa3 by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.  相似文献   

5.
Calmodulin is a ubiquitous Ca2+-binding protein, mediating the effect of Ca2+ on many enzyme systems and cellular reactions. Phospholipase A2 (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca2+ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A2 by Ca2+ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.  相似文献   

6.
The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in the range of 30–100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4–26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adynyah, J.T. Penniston and E. Carafoli, 1981, J.Biol.Chem.256,. 395 – 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that of the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.  相似文献   

7.
ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; Fsr) and soleus (slow skeletal muscle; Ssr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either Fsr or Ssr preparations. Fsr and Ssr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca2+; the exogenous addition of calmodulin (0.24 μM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca2+ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca2+ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca2+-ATPase was inhibited to a significant extent by 4880, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both Fsr and Ssr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca2+-transport remains to be determined.  相似文献   

8.
The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z >Ca4Z >Ca2Z ? CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10?7–10?6 M Ca2+, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10?7 – 10?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10?6 – 10?5 M.  相似文献   

9.
Calmodulin-like activity in the soluble fraction of Escherichia coli   总被引:8,自引:0,他引:8  
A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca2+-dependent cyclic AMP phosphodiesterase of Escherichiacoli. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca2+,Mg2+)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca2+-dependent fashion with an apparent Ka of 5 × 10?5M. The factor and brain calmodulin had no effect on the phosphodiesterase of E.coli. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.  相似文献   

10.
The human erythrocyte membrane Ca2+Mg2+ ATPase responded to the presence of an acidic phospholipase A2 and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the Ca2+M2+ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.  相似文献   

11.
A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid >collagen >thrombin >ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.  相似文献   

12.
Compound 4880, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 4880 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound 4880 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound 4880 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 4880 bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 4880 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound 4880 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 4880, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound 4880 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 4880 is proposed to be a promising tool for studying calmodulin-dependent processes.  相似文献   

13.
The concurrent effect of indomethacin or aspirin on prostaglandins (PGs) biosynthesis and on cellular fatty acid efflux were compared. Studies with rabbit kidney medulla slices and with isolated perfused rabbit kidney showed a marked difference between the two non-steroidal anti-inflammatory drugs, with regard to their effects on fatty acid efflux from kidney tissue. While aspirin effect was limited to inhibition of PGs biosynthesis, indomethacin also reduced the release of free fatty acids. In medullary slices, indomethacin inhibited the Ca2+ stimulation of phospholipase A2 activity and the resulting release of arachidonic and linoleic fatty acids. In the isolated perfused rabbit kidney, indomethacin inhibited the basal efflux of all fatty acids as well as the angiotensin II — induced selective release of arachidonate. Indomethacin also blunted the angiotensin II — induced temporal changes in the efflux of all other fatty acids. Neither indomethacin nor aspirin affected significantly the uptake and incorporation of exogenous (14C)-arachidonic acid into kidney total lipid fraction.Our tentative conclusion is that indomethacin inhibits basal as well as Ca2+ or hormone stimulated activity of kidney lipolytic enzymes. This action of indomethacin reduces the pool size of free arachidonate available for conversion to oxygenated products (both prostaglandin and non-prostaglandin types). The non-steroidal anti-inflammatory drugs can therefore be divided into two groups: a) aspirin-type compounds which inhibit PGs formation only by interacting with the prostaglandin endoperoxide synthetase and b) indomethacin-type compounds which inhibit PG generation by both reduction in the amount of available arachidonate and direct interaction with the enzyme.  相似文献   

14.
The influence of the neuroleptic trifluoperazine on the intracellular concentration of Ca2+ in macrophages of rats was studied using a Fura-2AM fluorescent Ca2+ probe. It was found that trifluoperazine causes a dose-dependent increase in the intracellular Ca2+ concentration associated with Ca2+ mobilization from intracellular Ca2+ stores and subsequent entry of Ca2+ into peritoneal macrophages of rats. It was also shown that inhibitors of phospholipase A2 (4-bromophenacyl bromide, prednisolone, and dexamethasone), cyclooxygenases (aspirin and indomethacin), and lipoxygenases (caffeic acid, zileuton, and baicalein) suppress Ca2+ responses induced by trifluoperazine in macrophages. The data obtained indicate the participation of enzymes and/or products of the cascade of arachidonic acid metabolism in the influence of trifluoperazine on the intracellular concentration of Ca2+ in peritoneal macrophages.  相似文献   

15.
When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved E.coli) in the presence of corticotropin, there was a Ca2+-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca2+ concentration. The addition of increasing Ca2+ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [14C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [14C]palmitic acid failed to release fatty acid in response to Ca2+, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca2+-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca2+-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca2+-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca2+-mediated activation of phospholipase A2 participates in the control of adrenocortical activity. However, since Ca2+ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca2+ on fatty acid release.  相似文献   

16.
The activity of protein kinase C as isolated and described by Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610–7616, can be markedly stimulated by Ca2+ in the presence of 4 mM Mg2+. This Ca2+ dependency does not require the presence of phospholipids or exogenous calmodulin. The increase in activity in the presence of Ca2+ is blocked by fluphenazine in the presence of 30 mM 2-mercaptoethanol. These results suggest that a calmodulin-like moiety may be a subunit of prokinase C.  相似文献   

17.
The effect of regucalcin, a novel Ca2+-binding protein, on Ca2+/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl2 and calmodulin. However, the activatory effect of both CaCl2 (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10-8 to 10-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl2 (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10-7 M). The inhibitory effect of regucalcin (10-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl2 (10 µM) and regucalcin (10-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.  相似文献   

18.
The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca2+ + Mg2+)-ATPase, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca2+ + Mg2+)-ATPase had an apparent half saturation constant of 77 ± 31 nM for free calcium, a maximum reaction velocity of 9.9 ± 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity (Ca2+ + Mg2+)-ATPase was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent Km of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity (Ca2+ + Mg2+)-ATPase. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity (Ca2+ + Mg2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Since the kinetic properties of the high-affinity (Ca2+ + Mg2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase, the high-affinity (Ca2+ + Mg2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.  相似文献   

19.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of (Ca2+ + Mg2+)-ATPase by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates (Ca2+ + Mg2+)-ATPase along a biphasic concentration curve (Km1 ≈ 1.4 μM, Km2 ≈ 330 μM), but in stripped membranes the curve is essentially hyperbolic (Km ≈ 7 μM). In calmodulin-bound membranes Ca2+ activates (Ca2+ + Mg2+)-ATPase at low concentrations (Km < 0.28 μM) in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein.  相似文献   

20.
The effects of copper on the activity of erythrocyte (Ca2+ + Mg2+)-ATPase have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca2+, calmodulin and (Mg-ATP)2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)2? against (Mg-ATP)2?Ki = 2.8 μM), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.  相似文献   

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