Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.

The gel-overlay technique with 125I-labelled calmodulin allowed the detection of several calmodulin-binding proteins of Mr 280 000, 150 000, 97 000, 56 000, 35 000 and 24 000 in canine cardiac sarcoplasmic reticulum. Only two calmodulin-binding proteins could be identified unambiguously. Among them, the 97 000-Mr protein that undergoes phosphorylation in the presence of Ca2+ and calmodulin, is likely to be glycogen phosphorylase. In contrast, the (Ca2+ + Mg2+)-activated ATPase did not appear to bind calmodulin under our experimental conditions. The second known calmodulin target is dephosphophospholamban, which migrates with an apparent Mr of 24 000. The dimeric as well as the monomeric form of phospholamban was found to bind calmodulin. Phospholamban shifts the apparent Kd of erythrocyte (Ca2+ + Mg2+)-activated ATPase for calmodulin, suggesting thus a tight binding of calmodulin to the proteolipid. Interestingly enough, phospholamban phosphorylation by either the catalytic subunit of cyclic AMP-dependent protein kinase or the Ca2+/calmodulin-dependent phospholamban kinase was found to inhibit calmodulin binding.     

Azidotyrosylcalmodulin derivatives. Specific probes for protein-binding domains

Azidocalmodulin has been shown to be a useful probe for calmodulin-binding proteins in a variety of systems (Andreasen, T. J., Keller, C. H. LaPorte, D. C., Edelman, A. M., and Storm, D. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2782-2785). In previous work, this calmodulin derivative was generated by the modification of lysyl residues. We report here that, on the basis of tryptic peptide mapping by reverse-phase high-pressure liquid chromatography, azidolysylcalmodulin prepared by these procedures is modified at 4 of the 7 lysines. We also report the preparation of azidocalmodulins in which the photolabile moiety is incorporated into a single known residue of the molecule by modifying one or the other of the two tyrosyl side chains. This yields azido-Tyr 99-calmodulin, with the photoaffinity label in Ca2+-binding loop III, and azido-Tyr 138-calmodulin, with the photoaffinity label in Ca2+-binding loop IV. The cross-linking characteristics of these two calmodulin derivatives show that the formation of a covalent adduct upon photolysis of a mixture of azidocalmodulin and a target protein is dependent on the location of the nitrene generated by the irradiation. Azido-Tyr 138-calmodulin shows a significant decrease in cross-linking efficiency to targets such as troponin I, troponin T, and myosin light-chain kinase, relative to azido-Tyr 99-calmodulin and the azidolysyl derivatives.     

Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca(2+)+Mg(2+)-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2mug/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca(2+)+Mg(2+)-stimulated ATPase by increasing the Ca(2+)-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca(2+)+Mg(2+)-dependent ATPase in addition to its effect on the Ca(2+)-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca(2+)+Mg(2+)-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca(2+) concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5mum-cyclic AMP inhibited Ca(2+)+Mg(2+)-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase. Secondly, trifluoperazine (20mum) antagonized the stimulation of Ca(2+)+Mg(2+)-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase by PKI.     

Topographical mapping of calmodulin-target enzyme interaction domains

Calmodulin derivatives, specifically biotinylated in domains I and III, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'-cyclic nucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-Mg2+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+2 binding region of domain III. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.     

Ca2+-ATPase activity in pancreatic acinar plasma membranes. Regulation by calmodulin and acidic phospholipids

A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.     

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.     

Conformation-sensitive modification of the type II calmodulin-dependent protein kinase by phenylglyoxal

Chemical modification by phenylglyoxal was used to investigate relationships between the structure, function, and regulation of the type II calmodulin-dependent protein kinase. Modification of the protein kinase by phenylglyoxal resulted in specific labeling of one distinct site, most likely an important arginine residue, with concomitant inactivation of the enzyme. Labeling and inactivation of the protein kinase was prevented by Mg2+-ADP which suggests that modification occurred at, or in close proximity to, its nucleotide-binding pocket. Half-maximal protection by Mg2+-ADP was enhanced by calmodulin which decreased the K0.5 for ADP from 540 to 61 microM. This response of the enzyme to calmodulin indicates that the modulator protein increases the affinity of the protein kinase for nucleotides. Inactivation of the enzyme by phenylglyoxal was dependent on the presence of Mg2+ or Ca2+/calmodulin, and further enhanced by the simultaneous addition of these effectors to the reaction. The Mg2+ effect is indicative of binding of this divalent metal ion to the protein kinase even in the absence of calmodulin and nucleotides. The stimulation of the modification reaction by calmodulin indicates an increase in the reactivity or accessibility of the modified residue in response to calmodulin-regulated conformational changes on the enzyme. The calmodulin-induced changes observed in this study may play important roles in the molecular mechanisms of activation of the type II calmodulin-dependent protein kinase.     

Regulation of erythrocyte Ca2+ pump activity by protein kinase C

Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

The inhibitor peptide of the mitochondrial F1.F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F1.F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the epsilon-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

Effect of calmodulin on myometrial cell membrane Ca2+,Mg2+-ATPase activity

Myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase purified by an affinity chromatography on calmodulin-sepharose 4B is calmodulin-dependent enzyme. Concentration of calmodulin required for half-maximal activation of enzyme was about 26 nM. By unlike to the enzymes originated from other tissues sensitivity to the calmodulin of the myometrial sarcolemma Ca(2+)-transporting ATPase was lower: calmodulin increased Vmax of ATPase about 1.25-fold, the apparent constant of the activation of enzyme by Ca2+ failed to alter independently on the phospholipid presenting at the enzyme isolation.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

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.     

Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.     

Effects of Al3+ on Ca2+-ATPase Activity on Chloroplasts of Rice and Relation to Calmodulin

The relationship between aluminium phytotoxicity and calmodulin has been studied with. calcium-dependent ATPase in chloroplasts of rice. This enzyme could be activated by extrinsic calmodulin. It showed that the activity of Ca2+-ATPase in chloroplasts was regulated by calmodulin. The activation of calmodulin to the enzyme might be inhibited by calmodulin antagonists, TFP and CPZ. The effects of A13+ on the activation of calmodulin was similar to that of the calmodulin antagonists. Calcium could reduce the inhibition of aluminiutn. It seems that there is a model of toxic responses in plants to aluminium: Al3+→calmodulin→target enzy mes→metabolism.     

Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C.

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.     

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.     

Interrelationships between thermal-, N-ethylmaleimide-, and Ca2+-calmodulin-mediated activation/inactivation of dynein ATPase activities

Interactive effects between calmodulin activation of 30 S dynein ATPase activity and activation by heat or N-ethylmaleimide (NEM) have been studied. Addition of calmodulin during the heat treatment caused a larger increment in ATPase activity (above that caused by heating alone) than did addition of calmodulin after the heat treatment. Similar results were obtained in experiments where activation was caused by NEM treatment. For both the heat and NEM treatments, the synergistic effect of calmodulin when present during the treatment was Ca²⁺ dependent although activation of ATPase activity by either treatment alone was not Ca²⁺ dependent. Heating 14 S dynein inhibited its ATPase activity and reduced the effectiveness of calmodulin as an activator. The activating effect of calmodulin added after heat or NEM treatments was about the same as if the calmodulin was present during the treatment, i.e., interactive effects were minimal. Concentrations of NEM that had little effect on the ATPase activity of 14 S dynein largely eliminated the ability of calmodulin to activate its ATPase activity. Chromatography of the heat-treated 14 S dynein on calmodulin-Sepharose 4B indicated that the loss of sensitivity of 14 S dynein ATPase to calmodulin was not due to loss of ability of the dynein to bind to calmodulin. Retention of calmodulin binding ability was also shown for heat-treated 30 S dynein. These results suggest that calmodulin and heat/NEM activate solubilized 30 S dynein ATPase by separate mechanisms which may include a common process.