Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes.

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.

Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.

Isolation of cardiac membrane proteolipids by high pressure liquid chromatography. A comparison of reticular and sarcolemmal proteolipids, phospholamban and calciductin.

Membrane-bound phosphorylatable proteolipids were reported to play a role in the regulation of transmembrane Ca2+ fluxes by catecholamines. A generally applicable purification procedure is described by which such proteolipids as the cardiac sarcoplasmic reticulum phospholamban is purified by solvent extraction followed by high pressure liquid chromatography on microparticulate silica. Phospholamban is thereby purified with a yield of 3.37 mg from 100 mg of sarcoplasmic reticulum proteins, significantly higher than that obtained by any of the previously reported procedures. It appeared homogeneous upon dodecyl sulfate-polyacrylamide gel electrophoresis where it is stained by Coomassie blue and detected by autoradiography. The same procedure is applicable to cardiac sarcolemmal calciductin. Both proteolipids exhibit the same Mr 11 000 and pI 3.7 upon two-dimensional gel electrophoresis. Their amino acid compositions are very similar if not identical. This raises the intriguing possibility that phospholamban and calciductin are identical though they obviously belong to different membranes.

Radioelectrophoresis: a specific microassay for parvalbumins. Application to muscle biopsies from man and other vertebrates.

A two-dimensional radioelectrophoretic method is described, by which parvalbumins from minute biopsy samples (approx. 50 mg) can be detected and quantitated by their 45Ca2+-binding properties. In the first dimension, parvalbumins are purified by sieving through a gradient polyacrylamide gel and collected at the bottom of the electrophoresis tubes. The second dimension is a disc electrophoresis in the presence of 45Ca2+. Parvalbumins can thus be identified and quantitated by three criteria: low molecular weight, acidic character and calcium-binding properties, since they are never exposed to denaturing conditions. Validity of the technique was demonstrated on carp myogen, and on extracts from rabbit psoas and heart muscles. Application of this method to the shrew fast beating myocardium shows that it does contain parvalbumin, in agreement with the proposed role of soluble relaxing factor (Pechère et al. (1977) FEBS Lett. 75, 111--1141. When applied to human muscle biopsies, radioelectrophoresis points to an uneven distribution of parvalbumin among different skeletal muscles. For the human limb muscles tested in this study, the parvalbumin content is similar to that of rabbit psoas muscle.

Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Isolation and characterization of sperm calmodulin.

Upon fluorescent staining with a goat antibody anti-ram testis calmodulin, washed bull sperm appears to contain calmodulin in the acrosome, in the post acrosomal region, in the neck region probably associated with the implantation plates and thin laminated fibers, and in a sheath around the upper part of the flagellum. Heads and midpieces + tails were separated by elutriation of sonicated sperm. Immunofluorescent labeling of fragments confirms the presence of calmodulin in implantation plates, where sonication disrupted heads from midpieces, and in a sheath around the midpiece and the upper part of the principal piece. These results were confirmed by electrophoretic and radioenzymatic assays of calmodulin in the fragments, using calmodulin-deficient Ca2+/calmodulin-dependent myosin light chain kinase. Small but significant amounts (approx. 3 micrograms per 10 (10) sperm) are found in midpieces + tails vs. approx. 280 micrograms in the same number of heads. These results are in agreement with a recent report from Jones et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2772-2776. Sperm calmodulin was purified from a whole sperm 1 M KCl extract and found to exhibit the same characteristics as other mammalian calmodulins isolated so far in terms of ultraviolet absorption spectrum and amino acid composition, including one residue of epsilon-N-trimethyllysine. Its behavior upon SDS-polyacrylamide gel electrophoresis was dependent on the presence or absence of Ca2+. The high performance liquid chromatography tryptic peptide maps were similar, if not identical, to mammalian calmodulin maps (Autric et al. (1980) Biochim. Biophys. Acta 631, 139-147). Sperm calmodulin is therefore probably identical to the somatic cell protein.

Regulation of calcium accumulation and efflux from platelet vesicles. Possible role for cyclic-AMP-dependent phosphorylation and calmodulin.

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.

The protein inhibitor of adenosine 3':5'-monophosphate-dependent protein kinases. Isolation and characterization of three isoinhibitors.

The protein inhibitor of adenosine 3':5 monophosphate-dependent protein-kinases has been purified from rabbit skeletal muscle by affinity chromatography on Sepharose-bound catalytic subunit of the kinase (Demaille et al. (1977) Biochemistry 16, 3080-3086). The inhibitory material can be separated into three species A, B and C either by polyacrylamide gel electrophoresis or by anion-exchange on DEAE-cellulose. However, the isoinhibitors display the same molecular weight and isoelectric point, and the same absence of acid-stable covalently bound phosphate. Their amino acid compositions are strikingly similar and their NH2-terminus are blocked. Also, their COOH-terminus appear to be very close to one another when studied by carboxypeptidase Y digestion. Their major difference lies in their inhibitory properties which are significantly weaker in inhibitor C (Ki = 0.87 nM) than in A and B (Ki = 0.18 and 0.23 nM, respectively). This is the first report on the existence of various forms of the protein-kinase inhibitor that exhibit different inhibitory properties and may play a role in the regulation of the protein-kinase system.

Calcium and magnesium binding by parvalbumin. A proton magnetic resonance spectral study.

Proton magnetic resonance spectroscopy has been used to monitor the kinetics and nature of the conformational transitions induced by binding of calcium and magnesium to carp parvalbumin. Rate constants have been determined for the exchange between the cation dependent conformational states of the protein in solution. These data enable a description of the kinetics and mechanism controlling the calcium flux in vivo during contraction.

Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction?

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation. A 4-step scheme for meiotic maturation.

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.

Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase.

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.