Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.     

Purification and some properties of a new Ca2+-binding protein (TCBP-10) present in tetrahymena cilium

A new Ca2+-binding protein, different from calmodulin, has been detected in the cilium and cell body of Tetrahymena. This protein, designated as TCBP-10, has been purified from the cells to homogeneity. TCBP-10 is an acidic protein (pI = 4.5) which shows a Ca2+-dependent mobility shift in alkali-glycerol-polyacrylamide gel electrophoresis. The protein is resistant to heat and trichloroacetic acid. The molecular weight of the protein is 10,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 22,000 by Sephadex G-50 gel filtration, suggesting that the native form of the protein is a dimer. The protein has a molar extinction coefficient of 6,500 at 282 nm. Equilibrium dialysis experiments revealed that the protein binds 1 mol of Ca2+/mol of protein with a dissociation constant of 27 microM. The protein contains a relatively large quantity of acidic amino acids, single residues of cysteine, histidine, and tryptophan, and no methionine. These properties are similar to those of some low molecular weight Ca2+-binding proteins belonging to the calmodulin family. Thus, the cilium of Tetrahymena contains a second Ca2+-binding protein in addition to calmodulin. We consider that TCBP-10 and calmodulin may play important cooperative roles in the Ca2+-regulation of ciliary movement in Tetrahymena.     

Binding of microtubule-associated protein 2 and tau to the intermediate filament reassembled from neurofilament 70-kDa subunit protein. Its regulation by calmodulin

Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to bind to the intermediate filaments reassembled from neurofilament 70-kDa subunit protein (= 70-kDa filaments). The binding was saturable. The apparent dissociation constant (KD) for the binding of MAP2 to the 70-kDa filaments was estimated to be 4.8 X 10(-7) M, and the maximum binding reached 1 mol of MAP2/approximately 30 mol of 70-kDa protein. The apparent KD for the tau binding was 1.6 X 10(-6) M, and the maximum binding was 1 mol of tau/approximately 3 mol of 70-kDa protein. It was also found that MAP2 and tau did not compete with each other for binding to the 70-kDa filaments. Most interestingly, calmodulin, a ubiquitous Ca2+-binding protein in eukaryotic cells, was found to inhibit the binding of MAP2 and tau to the 70-kDa filaments. The inhibition by calmodulin was regulated by changes in Ca2+ concentration around 10(-6) M, and was canceled by trifluoperazine, a calmodulin inhibitor.     

Effect of pH on Ca-binding properties of calmodulin and on its interaction with Ca-dependent phosphodiesterase of cyclic nucleotides

The fluorescence of dansyl immobilized on bovine brain calmodulin is sensitive to Ca2+. This effect is due to Ca2+ attachment to specific Ca2+-binding sites of calmodulin and is maintained within a wide range of pH. The native and dansyl-modified calmodulin preparations exert similar activating effects on Ca-dependent phosphodiesterase of cyclic nucleotides and have practically the same affinity for the enzyme. Using fluorescence measurements of the calmodulin--dansyl conjugate, it was shown that the decrease of pH from 9.0 down to 6.0 gradually decreases the constant of Ca2+ binding to calmodulin from 1.5 . 10(10) M-1 to 1.6 . 10(6) M-1. This decrease of pH does not affect the calmodulin affinity for phosphodiesterase. The activating effect of calmodulin on phosphodiesterase is more pronounced at acidic pH values (6.0-7.0) than at alkaline pH values (8.0-9.0).     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase

Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning,localization, and axonemal function of Tetrahymena centrin

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.     

Interaction of amiodarone and its analogs with calmodulin

Benzofurans have important actions on the electrical properties of myocardium; the biochemical basis of those actions is not known. Crystallographic examination of these compounds has revealed that benzofurans share structural homologies with the traditional calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene and trifluoperazine. In the present study, the ability of amiodarone, desethylamiodarone, and benziodarone to displace the fluorescent ligand 8-anilino-1-naphthalene sulfonic acid (ANS) from calmodulin, to modulate the fluorescence emission of dansylcalmodulin, and to inhibit the activation by calmodulin of bovine brain cyclic nucleotide phosphodiesterase and human erythrocyte membrane Ca2+-ATPase were investigated at concentrations ranging from 10(-8) to 10(-6) M. These benzofurans displaced ANS from calmodulin with nearly equal efficiency upon forming a 1:1 complex with that protein. Each of these compounds also produced a decreased fluorescence emission of dansylcalmodulin, but with relative efficiencies being desethylamiodarone greater than amiodarone greater than benziodarone. Amiodarone and desethylamiodarone inhibited calmodulin-stimulable phosphodiesterase activity with similar potencies. Amiodarone and benziodarone inhibited calmodulin-stimulable Ca2+-ATPase activity equally, but desethylamiodarone had no effect. The observed differential effects of the amiodarone analogs suggest that calmodulin may possess multiple benzofuran-binding sites that are recognized by specific targets and ligands of this Ca2+-binding protein and that the cellular action of amiodarone and its analogs may reflect calmodulin antagonism.     

Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.     

Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase

Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

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 microM 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 microM. 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 greater than Ca4Z greater than Ca2Z greater than or equal to 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 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is 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.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.     

Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase

Ca2+-regulated guanylate cyclase in ciliary membranes from Paramecium contained tightly bound calmodulin. Antisera against calmodulin from Tetrahymena and soybean inhibited enzyme activity. EGTA did not easily release calmodulin; however, La3+ inhibited guanylate cyclase by dissociation of calmodulin. While La could not replace Ca in the activation of guanylate cyclase, it substituted for Ca2+ in the activation of calmodulin-dependent phosphodiesterase from pig brain independently of whether homologous or Paramecium calmodulin was used. After removal of endogenous calmodulin from guanylate cyclase, reconstitution was achieved with calmodulin from Paramecium, Tetrahymena, pig brain, and soybean. Ca2+-binding proteins lacking trimethyllysine like calmodulin from Dictyostelium, parvalbumin, and troponin C failed to restore enzyme activity. The properties of the native and reconstituted guanylate cyclase/calmodulin complex were compared. Reassociation of calmodulin with its target enzyme was weak since all calmodulin remained in the supernatant after a single centrifugation. While most enzyme characteristics remained unchanged in the reconstituted complex, the inhibition by Ca greater than 100 microM was of a mixed-type compared to noncompetitive inhibition in the native enzyme. The regulation of the enzyme by cations was also altered. Whereas Ca was the most potent and specific activator of the native enzyme, in the reconstituted system Sr was far more effective.     

Identification and characterization of calmodulin-binding proteins in islet secretion granules

The binding of 125I-calmodulin to intact secretion granules and protein gel blots of secretion granules from pancreatic islet tissue was examined. Binding of 125I-calmodulin to intact secretion granules was Ca2+-dependent and inhibited by the calmodulin inhibitors trifluoperazine and calmidazolium. Binding was inhibited by excess (200 nM) unlabeled calmodulin, but not by parvalbumin, a Ca2+-binding protein which has little sequence homology to calmodulin. In order to study the binding of calmodulin to specific secretion granule proteins, secretion granules were solubilized, and the solubilized proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and incubated with 125I-calmodulin. Autoradiograms of the protein gel blots revealed the presence of three major calmodulin-binding proteins with approximate molecular weights of 73,000, 64,000, and 58,000. These proteins reversibly bound calmodulin in a calcium-dependent manner. Unlabeled calmodulin in the range of 0.1-1.0 nM competed with 125I-calmodulin for binding to these proteins, whereas troponin and parvalbumin were 100 and 1000-fold less effective, respectively. Trifluoperazine blocked binding to the granule proteins in a range of 10(-4) to 10(-5) M, and calmidazolium was effective between 10(-5) and 10(-6) M. Trypsin, at a concentration which did not lyse granules, markedly inhibited calmodulin binding to intact secretion granules. Protein blots from trypsin-treated granules showed that the three major calmodulin-binding proteins were absent. These results indicate that Ca2+-dependent calmodulin-binding proteins are present on the cytoplasmic surface of islet secretion granules and are consistent with the hypothesis that these proteins may play a role in secretion granule exocytosis.     

Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.