Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments

We have found that the 90-kDa heat shock protein (HSP90) prepared from a mouse lymphoma exists in homodimeric form under physiological conditions and has the ability to bind to F-actin (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A., in press). Here we show that calmodulin regulates the binding of HSP90 to F-actin in a Ca2+-dependent manner. The binding of HSP90 to F-actin occurred optimally under physiological solution conditions, i.e. in 2 mM MgCl2 + 100 mM KCl. The binding was saturable in a molar ratio of about 1 HSP90 (dimer) to 10 actins. HSP90 was dissociated from F-actin by the binding of tropomyosin to F-actin. Calmodulin was found to inhibit the binding of HSP90 to F-actin in a Ca2+-dependent manner. Moreover, the equilibrium gel filtration demonstrated that calmodulin binds to HSP90 in the presence of Ca2+, but not in the absence of Ca2+. These data indicate that HSP90 complexed with Ca2+-calmodulin is unable to bind to F-actin. Ca2+-dependent interaction of HSP90 with calmodulin as well as calmodulin-regulated binding of HSP90 to F-actin revealed here may provide new insight into the function of HSP90 and the regulation of actin structure in cells.     

Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin

Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Crossed immunoelectrophoresis of human platelet membranes. Effect of charge on association and dissociation of the glycoprotein GPIIb-GPIIIa membrane complex

The mechanism of association of the human platelet membrane GPIIb-GPIIIa-Ca2+ complex was studied by treating solubilized membranes with various enzymes and cationic peptides and by studying the binding of 45Ca2+ and 125I-fibrinogen before and after dissociation with EGTA and association with Ca2+. Neuraminidase shifted the complex cathodally (presumably due to cleavage of negatively charged domains), whereas trypsin had no such effect. The EGTA-dissociated complex was almost completely reassociated with neuraminidase or the cationic peptide, tetralysine. The monoclonal antibody 10E5, which specifically binds to the Ca2+-associated complex (not to its dissociated components), also bound to the neuraminidase-associated complex. Thus, Ca2+ is not necessary for the association of the complex. Neuraminidase treatment of washed intact platelets resulted in a cathodal shift of the membrane Triton X-100-extracted associated complex with no effect on its ability to dissociate in the presence of EGTA. Neuraminidase treatment of ADP-perturbed washed platelets also resulted in a cathodal shift of the associated complex; however, dissociation with EGTA was inhibited. Thus, critical neuraminidase-sensitive components of the complex (sialic acid residues) are not exposed on the surface of the platelet membrane of resting platelets, but do become accessible following platelet stimulation with ADP or membrane solubilization with Triton X-100. 45Ca2+ bound to the associated complex, to GPIIb of the dissociated complex (not to GPIIIa), to the Ca2+-reassociated complex, and to the neuraminidase-associated complex which had been dissociated with EGTA. Thus, neuraminidase-sensitive components of the solubilized membrane are not required for Ca2+ binding. 125I-fibrinogen bound to the associated complex (not the dissociated complex), to the Ca2+-reassociated complex, and to the neuraminidase-reassociated complex which had been dissociated with EGTA. Thus, Ca2+ is not necessary for 125I-fibrinogen binding to the major antigen complex.     

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

Interaction of calmodulin with troponin I and the troponin-tropomyosin-actin complex. Effect of Ca2+ and Sr2+ ions.

In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Control of actin filament length by phosphorylation of fragmin-actin complex

Fragmin is a Ca2(+)-sensitive F-actin-severing protein purified from a slime mold, Physarum polycephalum (Hasegawa, T., S. Takahashi, H. Hayashi, and S. Hatano. 1980. Biochemistry. 19:2677-2683). It binds to G-actin to form a 1:1 fragmin/actin complex in the presence of micromolar free Ca2+. The complex nucleates actin polymerization and caps the barbed end of the short F-actin (Sugino, H., and S. Hatano. 1982. Cell Motil. 2:457-470). Subsequent removal of Ca2+, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca2+ concentration. Here we report that this activity of fragmin-actin complex can be abolished by phosphorylation of actin of the complex. When crude extract from Physarum plasmodium was incubated with 5 mM ATP and 1 mM EGTA, the activities of the complex decreased to a great extent. The inactivation of the complex in the crude extract was not observed in the presence of Ca2+. In addition, the activities of the complex inactivated in the crude extract were restored under conditions suitable for phosphatase reactions. We purified factors that inactivated fragmin-actin complex from the crude extract. These factors phosphorylated actin of the complex, and the activities of the complex decreased with an increased level of phosphorylation of the complex. These factors, termed actin kinase, also inactivated the complex that capped the barbed end of short F-actin, leading to elongation of the short F-actin to long F-actin. Thus the length of F-actin can be controlled by phosphorylation of fragmin-actin complex by actin kinase.     

Ca2+ and calmodulin regulate microtubule-associated protein-actin filament interaction in a flip-flop switch

MAP2 (microtubule-associated protein 2) and tau factor are calmodulin-binding and actin filament-interacting proteins, respectively. We have examined the effect of Ca2+ and calmodulin on MAP-induced actin gelation by the low-shear falling-ball method, the high-speed centrifugation method, and electron microscopy using negative staining. Each MAP crosslinks actin filaments to increase the apparent viscosities and finally to form gels. Calmodulin inhibited MAP2- and tau factor-induced actin gelation (MAP2- and tau factor-actin interaction) only in the presence of Ca2+, but not in its absence. There were no differences in actin filament crosslinking activity of respective MAPs with or without Ca2+. MAP2 was not coprecipitated with F-actin only in the presence of Ca2+ and calmodulin determined by the high-speed centrifugation method. But MAP2 was found to bind to F-actin under any other conditions examined. In contrast, the tau factor-actin filament interaction could only be detected by the low-shear viscosity, but not by the high-speed centrifugation method. MAP2 and tau factor aggregated to form actin bundles as shown by electron microscopy. MAP2- or tau factor-induced bundle formation of actin filaments was inhibited only in the presence of Ca2+ and calmodulin, but not in the presence or absence of Ca2+. In conclusion, the interaction of MAP2- and tau factor-actin filaments is regulated by Ca2+ and calmodulin in a flip-flop switch.     

Calcium-sensitive modulation of the actomyosin ATPase by fodrin

Fodrin, a spectrin-like protein isolated from brain, is a long flexible molecule which binds calmodulin and cross-links F-actin. The effects of fodrin on the actin-activated ATPase of myosin have been examined. When added after ATP, fodrin inhibited the actomyosin ATPase. Two to three times as much fodrin was required for inhibition in the presence of Ca2+ as in its absence. Complete inhibition in the absence of Ca2+ occurred at about one fodrin to 200 actins. Inhibition does not appear to result from fodrin cross-linking F-actin, and, thereby, preventing the myosin filaments from reaching the actin filaments; but cross-linking may promote inhibition by trapping the myosin filaments within the cross-linked F-actin. When added before ATP, fodrin stimulated the actomyosin ATPase almost 3-fold in the presence of Ca2+ and by less than 50% in the absence of Ca2+. Stimulation is thought to result from fodrin cross-linking F-actin. After several minutes the stimulations in Ca2+ were greatly reduced, and in the absence of Ca2+ the actomyosin ATPases were substantially inhibited. Whether added before or after ATP, fodrin inhibited the actin-activated ATPase of myosin subfragment 1. This inhibition was also slightly Ca2+ sensitive.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

Ca2+-calmodulin-dependent regulation of F-actin-myelin basic protein interaction

Myelin basic proteins (MBP) interacts with F-actin resulting in the precipitation of a complex of both proteins. Electron microscope observations of this complex reveal the presence of ordered bundles of F-actin filaments similar to those obtained from F-actin and troponin I. In addition to the bundles, there also appear short fragments of F-actin filaments. In the presence of Ca2+ calmodulin causes a release of MBP from its complex with F-actin, accompanied by dissociation of F-actin bundles into separate filaments. Parallel to the binding of MBP to F-actin the ATPase activity of actomyosin is progressively reduced. This inhibition is reversed by calmodulin but only in the presence of Ca2+. Studies of the binding of S-1 to F-actin and to the F-actin-MBP complex indicate that the interaction sites for MBP and S-1 on the actin molecule are different.     

Metabolic, cationic and secretory response to D-glucose in depolarized and Ca(2+)-deprived rat islets exposed to diazoxide

D-glucose stimulates insulin release from islets exposed to both diazoxide, to activate ATP-responsive K+ channels, and a high concentration of K+, to cause depolarization of the B-cell plasma membrane. Under these conditions, the insulinotropic action of D-glucose is claimed to occur despite unaltered cytosolic Ca2+ concentration, but no information is so far available on the changes in Ca2+ fluxes possibly caused by the hexose. In the present experiments, we investigated the effect of D-glucose upon 45Ca efflux from islets exposed to both diazoxide and high K+ concentrations. In the presence of diazoxide and at normal extracellular Ca2+ concentration, D-glucose (16.7 mmol/l) inhibited insulin release at 5 mmol/l K+, but stimulated insulin release of 90 mmol/l K+. In both cases, the hexose inhibited 45Ca outflow. In the presence of diazoxide, but absence of Ca2+, D-glucose (8.3 to 25.0 mmol/l) first caused a rapid decrease in insulin output followed by a progressive increase in secretory rate. This phenomenon was observed both at 5 mmol/l or higher concentrations (30, 60 and 90 mmol/l) of extracellular K+. It coincided with a monophasic decrease in 45Ca efflux and either a transient (at 5 mmol/l K+) or sustained (at 90 mmol/l K+) decrease in overall cytosolic Ca2+ concentration. The decrease in 45Ca efflux could be due to inhibition of Na(+)-Ca2+ countertransport with resulting localized Ca2+ accumulation in the cell web of insulin-producing cells. A comparable process may be involved in the secretory response to D-glucose in islets exposed to diazoxide and a high concentration of K+ in the presence of extracellular Ca2+.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

Sidedness of K+ activation of calcium transport in the reconstituted sarcoplasmic reticulum calcium pump

Sidedness of the effect of K+ on Ca transport by the sarcoplasmic reticulum Ca pump reconstituted into soybean phospholipid vesicles was investigated. The reconstituted vesicles which sustained a high rate of Ca transport even in the absence of Ca-precipitating anions exhibited low passive permeabilities to 42K+, 86Rb+, or 45Ca2+. Evidence was presented that K+ activated the Ca pump on the external surface of the vesicles and that it was not taken up by the vesicles during the pump activity. In the presence of high externally added K+, the reconstituted vesicles preloaded with K+ exhibited a significantly higher Ca transport activity than the vesicles preloaded with Tris+ but not the ones preloaded with Li+. Ca transport by the K+-loaded vesicles was accompanied by a small amount of K+ efflux, which corresponded to about 20% of the amount of Ca+ taken up. Since the intravesicular K+ did not affect the turnover of the ADP-insensitive component (E2P) of the phosphoenzyme intermediate formed during the pump cycle, it was concluded that the intravesicular K+ stimulated the Ca pump activity indirectly by compensating the charge imbalance caused by the electrogenic Ca2+ movement. These results thus indicate that K+ activates the Ca pump only on the cytoplasmic side of the sarcoplasmic reticulum membrane, but it is not obligately transported across the membrane under conditions where K+ fully activates the Ca pump.     

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets

The present study aimed at comparing the effects of glucose on ionic and secretory events in freshly isolated and 5-7 day cultured rat pancreatic islets. The capacity of glucose to provoke insulin release was severely reduced in islets maintained in culture. Whether in freshly isolated or cultured islets, glucose provoked a marked and sustained decrease in 45Ca2+ outflow from islets deprived of extracellular Ca2+. In the presence of extracellular Ca2+ throughout, the magnitude of the glucose-induced secondary rise in 45Ca2+ outflow was reduced in cultured islets. Glucose provoked a weaker increase in [Ca2+]i in islet cells obtained from cultured islets than in islet cells dissociated from freshly isolated pancreatic islets. On the other hand, the stimulatory effect of carbamylcholine on 45Ca2+ outflow was unaffected by tissue culture. Lastly, in islet cells obtained from cultured islets, the increase in [Ca2+]i evoked by K+ depolarization averaged half of that observed in control experiments. These results indicate that the reduced secretory potential of glucose in cultured pancreatic islets can be ascribed to the inability of the nutrient secretagogue to provoke a suitable increase in Ca2+ influx.     

Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions

Extracts of human platelets contain a 90,000-Da protein that is retained by DNase I-agarose in the presence of Ca2+. The 90-kDa protein, tightly complexed with platelet actin, can be eluted from DNase I-agarose by ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA). The platelet 90-kDa protein is immunologically related to rabbit macrophage gelsolin. The 90-kDa protein-actin complex was purified from platelet extracts using DEAE-Sephacel, Sephadex G-200, and hydroxyapatite and is stable in EGTA and 0.8 M KCl. The purified complex will modulate the assembly of fluorescently labeled 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole-actin in the presence of both Ca2+ and EGTA. In addition, the complex affects the low shear viscosity of F-actin solutions in the presence of both Ca2+ and EGTA. Finally, the complex increases the critical concentration for actin assembly about 4-fold. The results are consistent with a strong preferential binding to or capping of the barbed end of actin filaments by the complex in either Ca2+ or EGTA.     

Myosin may stay in EADP species during the catch contraction in scallop smooth muscle

Myosin (opaque myosin) isolated from the opaque portion of scallop smooth muscle, a catch muscle, was subjected to limited digestion by trypsin during the steady-state ATPase reaction. The 200-kDa heavy chain of opaque myosin was cleaved into 125- and 74-kDa fragments. The proteolytic rate in the absence of Ca2+ was lower than that in the presence of Ca2+, and was similar to that in the presence of ADP and absence of Ca2+. The results suggest that the steady-state intermediate of opaque myosin ATPase in the absence of Ca2+ is EADP, which is consistent with the previous results based on the difference UV-absorption spectrum (Takahashi, M., Sohma, H., & Morita, F. (1988) J. Biochem. 104, 102-107). In the presence of F-actin, the proteolytic rates were decreased, but the digestive patterns by trypsin were similar to those of myosin alone. Even in the presence of F-actin, the proteolytic rate during the ATPase reaction in the absence of Ca2+ was lower than that in the presence of Ca2+, and was similar to that in the presence of ADP and absence of Ca2+. In addition, there was another trypsin-susceptible site which is probably located at 18 kDa from the N-terminal of the heavy chain. The site in the absence of Ca2+ was hardly cleaved when ATP or ADP was present. Similar tendencies were observed even in the presence of F-actin. These findings suggest that the intermediate of opaque myosin ATPase at the steady state in the absence of Ca2+ is EADP even in the presence of F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)