Possible role of calmodulin in the control of histamine release from human basophil leukocytes

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.     

Effects of calmodulin antagonists on auxin-stimulated proton extrusion in Avena sativa coleoptile segments

The effect of the 5 calmodulin (CaM) antagonists trifluoperazine (TFP). compound 48/80, N-(6-aminohexyl)-naphthalenesulfonamtde (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium on auxin-dependent medium acidification was investigated in abraded segments of Avena sativa L. cv. Victory I. Buffering capacity, Asn content, and changes in pH of bathing solutions were measured in the presence of these inhibitors. When coleoptiles were treated with TFP or compound 48/80, the Asn content and the buffering capacity increased, thus suggesting that plasma membrane permeability was modified. On the contrary. the effect of calmidazolium, W-5. and W-7 on Asn release and buffering capacity was rather low; only small effects being observable at the highest concentration employed. Calmidazolium and W-7 strongly inhibited auxin-dependent medium acidification. W-5 did not affect medium acidification. The specificity of these CaM antagonists and their effects on medium acidification are discussed. The data adduced is consistent with the working hypothesis which postulates an essential role for the Ca²⁺-CaM system on auxin-dependent medium acidification.     

Stage Depandent Inhibition of Plasmodiun falcipaium falciparum by Potent Ca2+ and Calmodulin Modulators

The effects Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 μM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 μM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, ditiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythocyte invasion by merozoites. Fluroscence microscopy with the cationic flurescent dye rhodamine 123 revealed that nicardipine. TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although al Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Stage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators

The effects of Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 microM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 microM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, diltiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythrocyte invasion by merozoites. Fluorescence microscopy with the cationic fluorescent dye rhodamine 123 revealed that nicardipine, TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although all Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.     

Calmodulin antagonists inhibit secretion in Paramecium

Secretion in Paramecium is Ca2+-dependent and involves exocytic release of the content of the secretory organelle, known as the trichocyst. The content, called the trichocyst matrix, undergoes a Ca2+-induced reordering of its paracrystalline structure during release, and we have defined three stages in this expansion process. The stage I, or fully condensed trichocyst, is the 4 microns-long membrane-bounded form existing prior to stimulation. Stage II, the partially expanded trichocyst, we define as an intermediate stage in the transition, preceding stage III, the fully expanded extruded form which is a 20-40 microns-long needlelike structure. These stages have been used to assay the effects of trifluoperazine (TFP) and W-7, calmodulin (CaM) antagonists, on trichocyst matrix expansion in vivo. TFP and W-7 are shown to reversibly block matrix release induced by picric acid. Ultra-structural examination reveals that one effect of this inhibition is reflected in the organelles themselves, which are prevented from undergoing the stage I-stage II transition by preincubation in 14 microM TFP or 35 microM W-7 before fixation. This inhibition of expansion by TFP can be moderated but not abolished by high extracellular Ca2+ (5 mM). The moderation by high Ca2+ can be eliminated by raising TFP concentration to 20 microM. A possible explanation for the ability to titrate the inhibition in this manner is that TFP is acting to block expansion by binding to the Ca2+-CaM complex. Brief exposure of cells to the Ca2+ ionophore A23187 and 5 mM Ca2+ following TFP treatment promotes matrix expansion, although in 14 microM TFP a residual level of inhibition remains. These results suggest that, following stimulation, CaM regulates secretion in Paramecium, possibly by controlling the Ca2+-dependent matrix expansion which accompanies exocytosis in these cells.     

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.     

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.     

Purified red blood cell Ca-pump ATPase: Evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca²⁺-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca²⁺-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca²⁺-pump ATPase and/or its lipid environment is suggested.     

Involvement of calmodulin and calmodulin binding site in the TSH receptor of thyroid

Calmodulin (CaM) antagonists, W-7 and W-5, chlorpromazine and haloperidol, especially W-7 inhibited 125I-bovine TSH binding to human and porcine thyroid receptors dose-dependently in the presence of calcium ion. This inhibitory effect of W-7 was diminished by the addition of ethylenglycol tetraacetic acid (EGTA) or ethylendiamine tetraacetic acid (EDTA). CaM also dose-dependently inhibited the binding of 125I-TSH to thyroid receptor in the presence of calcium ion. TSH binding to thyroid receptor was completely inhibited by more than 30 micrograms of CaM, and this inhibition was abolished by adding EGTA. On the other hand, the antibody to CaM inhibited partially 125I-TSH binding to its receptor. These results suggest involvement of CaM and CaM-binding sites in the TSH receptor of thyroid.     

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K_d) of 6.5 μM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z′ factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

ＳｉｎｃｅｔｈｅｆｉｒｓｔｒｅｐｏｒｔｂｙＢｅｎｎｅｔｔ[1]ｔｈａｔｍｕｌｔｉｐｌｅｃｈｌｏｒｏｐｌａｓｔｐｒｏｔｅｉｎｓｃｏｕｌｄｂｅｐｈｏｓｐｈｏｒｙｌａｔｅｄｂｙａｎｅｎｄｏｇｅｎｏｕｓｋｉｎａｓｅｗｈｉｃｈｗａｓｓｔｉｍｕｌａｔｅｄｂｙｌｉｇｈｔａｎｄｒｅｄｕｃｉｎｇａｇｅｎｔｓ,ｍｏｓｔｉｎｖｅｓｔｉｇａｔｉｏｎｓｒｅｇａｒｄｉｎｇｆｕｎｃｔｉｏｎａｎｄｒｅｇｕｌａｔｉｏｎｏｆｔｈｙｌａｋｏｉｄｐｒｏｔｅｉｎｐｈｏｓｐｈｏｒｙｌａｔｉｏｎｈａｖｅｂｅｅｎｃｏｎｃｅｎｔｒａｔｅｄｏｎｔｈｅ…     

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.     

Calmodulin regulation of cholinergic muscarinic receptor: effects of calcium and phosphorylating states

Calmodulin (CaM) regulation of cholinergic muscarinic receptor was investigated using synaptic membrane isolated from rat brains and [3H]-QNB as a binding ligand. CaM exerts a biphasic effect on receptor binding showing both a Ca2+-dependent receptor loss and an increase depending on the state of membrane phosphorylation. Calcineurin, a CaM-dependent protein phosphatase, mimicked the stimulatory effect of CaM in a dose-dependent manner. CaM-antagonists, W-7 and TFP reversed the stimulatory effect by CaM. A mechanism of protein phosphorylation and dephosphorylation of the cholinergic muscarinic receptors regulated by CaM-Ca2+ was proposed.     

Trifluoperazine and W-7 inhibit mating in Chlamydomonas at an early stage of gametic interaction

Gametic mating by Chlamydomonas reinhardi is inhibited in a dose-dependent and reversible manner by the calmodulin antagonists trifluoperazine (TFP) and W-7, but not by W-5, an analog of W-7 having lower affinity for calmodulin. Quantitation of the sequential steps of mating showed that TFP and W-7 both allow normal levels of flagellar agglutination but prevent all subsequent steps. Gametes agglutinate aberrantly and do not form mating pairs. Further, both of these drugs prevent the translocation of latex beads along the flagellar surface. Our observations suggest that calmodulin may play an integral role in the translocation of flagellar adhesion sites during the tip-locking stage of the Chlamydomonas mating reaction. Flagellar surface motility may be crucial to the transduction of signals during mating and may share regulatory mechanisms with other forms of surface motility.     

Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture.

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.     

Neuroprotective effects of a dihydropyridine derivative, 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarbox ylic acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester (CV-159), on rat ischemic brain injury

CV-159, 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic++ + acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester, is a dihydropyridine derivative that blocks the L-type Ca2+ channel and inhibits the calmodulin (CaM)-dependent pathway. In this study, we examined the effects of CV-159 on rat ischemic brain injury. CV-159 (5 and 10 mg/kg, p.o.) gave significant protection against delayed neuronal death in the hippocampal CA1 region after 15-min transient forebrain ischemia. In contrast, the Ca2+ antagonists nicardipine (1 and 10 mg/kg, p.o.) and nifedipine (1 mg/kg, i.p.) and the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, 500 ng, i.c.v.) had no effect on this hippocampal neuronal death. CV-159 also diminished the size of the brain infarct after permanent middle cerebral artery (MCA) occlusion, although physiological variables, including regional cerebral blood flow, were not affected. The increase in the water content of the infarcted cortex induced by MCA occlusion was significantly reduced by CV-159. On the other hand, neither nicardipine nor nifedipine affected the brain infarct size, volume or increased water content induced by MCA occlusion, as previously reported (A. Sauter and M. Rudin, Am. J. Hypertens. 4 121S-127S, 1991). These findings indicate that Ca2+ antagonists, such as nicardipine and nifedipine, and W-7 have no effect on rat ischemic brain injury. The results suggest that CV-159 protects against ischemic brain injury. This might be mediated by both blocking the L-type Ca2+ channel and inhibiting CaM-dependent function via Ca2+/CaM binding at a different binding site from that of W-7 to CaM (H. Umekawa, K. Yamakawa, K. Nunoki, N. Taira, T. Tanaka, and H. Hidaka, Biochem. Pharmacol. 37 3377-3381, 1988).     

钙调素对钙调素结合蛋白-10和玉米非特异性脂转移蛋白与脂质结合活性的影响

荧光标记的脂质结合实验表明,钙调素结合蛋白-10（CaMBP-10）具有典型的植物非特异性脂质转移蛋白与脂质结合的特性。进一步实验研究了钙调素（calmodulin,CaM）对CaMBP-10和玉米nsLTP与脂质结合的活性的影响,结果显示无论在有钙和无钙条件下,CaM对两者的影响均有不同之处,W-7和TFP能消除CaM的影响。提示CaM不仅与CaMBP-10和玉米nsLTP特异性相互作用,而且对2种脂转移蛋白可能具有不同的调节机制。     

Ca/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca²⁺/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.