Calmodulin Concentration Research Articles

Decrease in calmodulin concentrations during heparin-induced capacitation in bovine spermatozoa.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

Higher Ca2+ sensitivity of triton-skinned guinea pig mesenteric microarteries as compared with large arteries.

Small mesenteric resistance arteries and the main branch of the mesenteric artery (outer in situ diameter 115 +/- 3 microns [n = 76] and greater than 1,000 microns, respectively) were skinned with 1% Triton X-100. Both preparations were mounted as rings for circumferential force measurement in an EGTA solution (free Ca2+, less than 10 nM; calmodulin, 0.3 microM; pH 6.7). Force-pCa curves were obtained by increasing free Ca2+ (0.05 to 30 microM). The resulting dose-dependent contractions, after normalization to maximal force development (arteries, 21.4 +/- 2.4 [n = 3]; arterioles, 15.3 +/- 2.1 mN/mm2 [n = 5]) were fitted to sigmoidal force-pCa curves. Values of ED50 and of the cooperativity factor h were 6.08 and 2.39 in arterioles and 5.64 and 1.64 in arteries. The higher Ca2+ sensitivity of arteriolar preparations remained at pH 7.0 at higher calmodulin concentrations and after inhibition of smooth muscle phosphatase with okadaic acid. Total myosin light chain kinase activity in crude arteriolar extracts (using [gamma-32P] ATP and isolated gizzard light chains as substrates) was approximately 25% of arterial kinase. Both kinase preparations had identical Ca2+ sensitivities. Likewise, total arteriolar phosphatase activity (using 32P-labeled gizzard light chains) was approximately 25% of the arterial activity; both phosphatases had an identical sensitivity toward okadaic acid. The ratio of kinase/phosphatase activities was identical in both tissues. Extracts of both tissues contained two isozymes of the myosin heavy chain as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca2+ (50 microM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 microM); the inhibitory effect was complete at 1.0 microM. Regucalcin (1.0 microM) did not have an appreciable effect on basal activity without Ca2+ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160-480 U/ml). However, regucalcin (1.0 microM) did not inhibit Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 microM Ca2+ added. Meanwhile, Cd2+ (25-100 microM)-induced decrease in Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 microM). The present results suggest that regucalcin can regulate Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca2+ in liver cells.

Developmental changes in phosphorylation of MAP-2 and synapsin I in cytosol and taxol polymerised microtubules from chicken brain

In cytosol, cyclic AMP stimulated phosphorylation of microtubule associated protein-2 (MAP-2) increased from 2 days to adult in proportion to the increase in the concentration of MAP-2. By contrast, the calmodulin stimulated phosphorylation of MAP-2 decreased in proportion to the decrease in the concentration of calmodulin stimulated protein kinase II (CMK II). Similarly, the cAMP stimulated phosphorylation of the site on synapsin I labeled by the cAMP stimulated protein kinase (PKA) changed little during development whereas the calcium/calmodulin stimulated phosphorylation of the CMK II site decreased dramatically in proportion to the decrease in the concentration of CMK II. The decrease in the concentration of CMK II which occurs in cytosol during synapse maturation was also observed in taxol polymerised microtubules and the effects of the change in the relative concentrations of CMK II and PKA on the phosphorylation of MAP-2 and synapsin I in this fraction were similar to that observed in the cytosol. These results are consistent with the hypothesis that the developmental changes in phosphorylation of endogenous substrates by PKA is controlled largely by changes in the concentration of those substrates, whereas the concentration of CMK II is limiting so that the developmental changes in the phosphorylation of endogenous substrates by CMK II are a function of the concentration of CMK II itself as well as the concentration of endogenous substrates. Some possible functional consequences of this during synapse maturation are discussed.

Calmodulin-dependent protein kinase II. Kinetic studies on the interaction with substrates and calmodulin

The kinetic reaction mechanism of calmodulin (CaM)-dependent protein kinase II (CaM-kinase II), including the regulatory mechanism by CaM, was studied by using microtubule-associated protein 2 (MAP2) as substrate under steady-state conditions. The detailed kinetic analyses of the phosphorylation of MAP2 and its inhibitions by the reaction products and by an ATP analogue, 5′-adenylylimidodiphosphate, revealed the rapid-equilibrium random mechanism. In the absence of Ca 2+, CaM-kinase II was inactivated by incubation with ATP. The inactivation rate was dependent on the concentrations of ATP and MAP2, suggesting that these substrates can bind to the enzyme even in the absence of Ca 2+/CaM. The activation of the enzyme by CaM reached the maximum when about 10 mol of CaM bound to 1 mol of CaM-kinase II, indicating the stoichiometry of the binding of one CaM to one subunit of the enzyme. The enzyme activity as a function of the concentration of CaM showed a sigmoidal curve. The concentration of CaM required for the half-maximal activation was dependent on the concentration of ATP at a fixed concentration of MAP2, although the Hill coefficient was unaffected by the concentration of ATP. A possible reaction mechanism of CaM-kinase II, including the phosphorylation of MAP2 by the enzyme and the binding of CaM to the enzyme, is discussed.

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10(-5) M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 microM compared to 1 microM of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2(+)-binding domain with complete helix-loop-helix structure ("EF hand"). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.

Regulation of red cell membrane deformability and stability by skeletal protein network

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.

Temporal relationship between force, ATPase activity, and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration [( Ca2+]) was increased from pCa (-log[Ca2+]) 9 to pCa 4.5 at low calmodulin concentration (0.05 microM), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 microM), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting crossbridges, but may be maintained by dephosphorylated crossbridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.

Invasive adenylyl cyclase of Bordetella pertussis. Physical, catalytic, and toxic properties.

A rapid two-step purification to homogeneity of the calmodulin-activated adenylyl cyclase from urea extracts of Bordetella pertussis organisms (strain 114) is described. Catalytic and invasive activities are purified 30- and 177-fold, respectively, and virtually no degraded forms are found. Specific activities are 0.4 mmol/min/mg and 0.5 mumol/mg of enzyme protein/mg of cell protein/min for catalytic and invasive activities, respectively. The 15 amino-terminal amino acids agree with those deduced from the DNA sequence, as does the molecular mass of 175 kDa (guanidine) or 177 kDa (urea) obtained by equilibrium sedimentation. The larger apparent molecular mass seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be ascribed to anomalous migration. Half-maximal cyclase activation occurs at 3-4 X 10(-10) M calmodulin in the presence of Ca2+ and at 2 X 10(-8) M calmodulin in its absence. Ca2+ activation is maximal at 60-100 microM free CaCl2 (at low calmodulin concentrations), and free Ca2+ concentrations above approximately 125 microM are inhibitory at any calmodulin concentration. Extracellular Ca2+ is essential for intoxication. In Chinese hamster ovary cells, exogenous calmodulin does not inhibit penetration of the cyclase.

Structurally distinct mammalian calmodulins

Rabbits immunized with dinitrophenylated calmodulin produced monospecific antibody against CaM. Using the purified antibody, enzyme-linked immunosorbent assays (ELISAs) were carried out for calmodulin (CaM). The immunological heterogeneity of human and bovine CaMs from erythrocytes was investigated by means of indirect and inhibition ELISAs. The cross-reactivity between the two CaMs was found to be 50% in the indirect ELISA. An eight times higher concentration of human CaM was necessary to produce 30% inhibition in an inhibition assay. The effect of anti-bovine CaM on the stimulation of the red cell membrane calcium pump by human and bovine CaM has also been studied. We have found that (a) the human and bovine CaMs showed indistinguishable activator activities; (b) the antibody partially inhibited the stimulating effects of CaMs; (c) the inhibition was much less effective in the case of stimulation by human CaM. These results suggest that there is a difference in the reactivity of the anti-bovine CaM antibody with bovine and human CaMs. This difference can be attributed to a slight deviation in the antibody binding structure of the two mammalian CaMs in or near the antigenic site of the CaMs.

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase

The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.

Effects of ruthenium red on activation of Ca 2+-dependent cyclic nucleotide phosphodiesterase

Ruthenium red inhibited Ca 2+-dependent phosphodiesterase (Ca 2+-PDE) selectively with an IC 50 value of 15 μM. Increasing calmodulin concentration in the presence of both 100 μM and 4000 μM Ca 2+ completely reversed the inhibition of Ca 2+-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca 2+-PDE activity was also overcome by increasing the concentration of Ca 2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca 2+-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.

Phosphorylation of smooth muscle myosin light chain kinase by Ca 2+/calmodulin-dependent protein kinase II: Comparative study of the phosphorylation sites

Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca 2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 ± 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 ± 0.05 and almost completely inhibited phosphorylation of sites in two peptides ( 32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the apparent affinity of MLC-kinase for CaM. Binding of CaM to MLC-kinase inhibited the phosphorylation to 0.84 ± 0.13. Complete tryptic digests contained two major 32P-peptides as reported previously. One of the peptides, whose phosphorylation was inhibited in the presence of excess calmodulin, appeared to be the same as P2. Automated Edman sequence analysis suggested that both CaM-kinase II and cAMP-dependent protein kinase phosphorylated this peptide at the second of the two adjacent serine residues located at the C-terminal boundary of the CaM-binding domain. However, the other peptide phosphorylated by cAMP-dependent protein kinase, regardless of whether CaM was bound, was different from P1 and P3. Thus, MLC-kinase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of CaM-kinase II and is blocked by Ca 2+/CaM-binding.

Activated state in the lepidocrocite structure during thermal treatment

change of AMP against ATP and of calcium-flee calmodulin against calcium-loaded calmodulin the ATPase complex is workable again. Obviously, the energy transfer via calcium has the advantage that in case of emergency needs great amounts of free energy can be made available in a short time independently of the ATP supply. This is done by. flushing calcium-free calmodulin produced by continuous pumping and stored in the nearly calcium-flee cytosol with calcium coming from the periphery or any other calcium source of the cell. Thus the calcium/calmodulin system is part of a storage power station of the cell. In the pumping process based on the calcium transfer by calmodulin the lowest calcium concentration is set by the spontaneous dissociation of the calcium/calmodulin complex. This is consistent with the assumption made above to evaluate the upper limit of the free energy production by the calcium-loading of calmodulin. If a sustained stimulation results in a sustained reaction of the cell, the detour, via the cytosol, in the transport of calcium-loaded and calcium-flee calmodulin between the adenylate cyclase complex and the ATPase complex may be avoided. So the calcium cycling in the membrane region, described in [4] will be established. An increase in the concentration of calcium-loaded calmodulin in the cytosol not only starts the pumping process mentioned above but also activates phosphodiesterase which, as is well known, destroys cAMP by hydrolysis [2]. The main difference in the new concept as compared to the former models is seen in the fact that it attributes to calcium not only the function of a messenger but also the function of a transporter for the free energy needed to start endergonic reactions. The transfer of free energy by calcium does not need to be restricted to the production of cAMP for the initiation of chemical reactions. It can be expected that also for other endergonic processes in the cell, e.g., for the generation of mechanical work the transfer of free energy by calcium is of importance.

Calmodulin concentration in mucus of rainbow trout,Salmo gairdneri, exposed to combinations of acid, aluminum, and calcium

As a result of increasing acidification in various watersheds elevated levels of aluminum have been observed in soil and surface water. The toxicity of Al to fish has been shown to be positively correlated with the concentration of inorganic monomeric. The exact mechanism(s) of Al toxicity is not fully understood. Recently, the presence of calmodulin (CaM), a calcium-regulating protein, has been reported in fish gills and mucus. Calmodulin selectively binds inorganic monomeric Al causing conformational changes in the protein. Aluminum-induced conformational changes cause a reduction in the ability of calmodulin to mediate Ca-dependent phosphodiesterase and ATPase activity. Calmodulin also plays a key role in coordinating the effects of secondary messenger systems in response to cellular stimulation. Given the involvement of calmodulin in numerous biochemical pathways, its interaction with aluminum may be a key lesion in the broadly defined syndrome of aluminum toxicity. The present study was undertaken to establish a relationship between Al concentration in aqueous solution and the quantity and activity of CaM in the mucus of adult rainbow trout (Salmo gairdneri). Fish were exposed to various levels of pH, Ca, and Al. Mucus was collected and the amount of CaM was determined. The ability of the Al-exposed CaMmore » to activate the phosphodiesterase enzyme system was also evaluated.« less

Inhibition by new anthraquinone compounds, K-259-2 and KS-619-1, of calmodulin-dependent cyclic nucleotide phosphodiesterase

K-259-2 and KS-619-1, novel anionic anthraquinone metabolites isolated from culture broth of microorganisms, inhibited activation of bovine brain phosphodiesterase induced by calmodulin (CaM), sodium oleate, or limited proteolysis with almost equal potency. The inhibition of calmodulinactivated phosphodiesterase (CaM-PDE) by K-259-2 or KS-619-1 was overcome by a higher concentration of CaM. Direct interaction of K-259-2 and KS-619-1 with CaM was confirmed through use of hydrophobic fluorescent probes. Kinetic analysis revealed that the inhibition of the trypsin-activated phosphodiesterase was competitively inhibited by K-259-2 or KS-619-1 with respect to cAMP. Addition of a lower amount of either phosphatidylserine or sodium oleate to the reaction mixture was efficacious in attenuating the inhibition of the CaM-PDE by W-7, chlorpromazine, trifluoperazine, compound 48 80 , or R-24571 but, in contrast, had little or no effect on the inhibition by K-259-2 or KS-619-1. In conclusion, K-259-2 and KS-619-1, unlike so-called CaM antagonists, do not interact with phosphatidylserine or sodium oleate and it appears that these novel anthraquinone compounds inhibit the enzyme not only via CaM antagonism but possibly also by interacting directly with the enzyme.

The calmodulin-protein-kinase system of Mytilus edulis catch muscle

1. 1. The Ca 2+-dependent modulator protein calmodulin (CaM) and a calmodulin-dependent protein kinase (CaM kinase) were isolated and characterized from catch musles of the bivalve Mytilus edulis L. 2. 2. CaM was purified to homogeneity from the heat-denatured supernatant of the muscles by DEAE-cellulose chromatography. The overall purification was estimated to be 30,000-fold. It was identified as calmodulin (CaM) on the basis of its ability to activate CaM-deficient PDE and CaM kinase in presence of Ca 2+, its inhibition by trifluoperazine and its apparent mol. wt of 15,900. 3. 3. The concentration of CaM in the muscle tissues of the mussel was about 10 times as high as in non-muscle tissues. 4. 4. Calmodulin kinase was purified from myofibrils of the catch muscles by a three step procedure involving chromatography on Affi-Gel Blue, Mono Q and Superose 6 columns. The overall purification was ca 5000-fold. Calmodulin kinase is a monomeric enzyme with an apparent mol. wt of 36,000. 5. 5. The activity of CaM kinase is absolutely dependent on Mg 2+. The Ca 2+-sensitivity of the Superose 6 preparation is absolutely dependent on external CaM, while the Mono Q preparation still contains CaM. The addition of CaM to the CaM-deficient kinase in a mole to mole ratio activates the enzyme 5-fold. 6. 6. The CaM kinase displays a narrow protein specificity. Its favorite substrate was molluscan paramyosin. It incorporates 4 moles 32P/mole paramyosin at 10 −5 moles Ca 2+/l. The K m for paramyosin is 2.3 μmoles/l. 7. 7. The CaM kinase itself seems to be the substrate of a cAMP kinase present in kinase preparation eluted from Mono Q columns.

Purification and characterization of Tora-mame (Phaseolus vulgaris) seed calmodulin.

We purified calmodulin to apparent homogeneity from Tora-mame (Phaseolus vulgaris) seeds, one of the Japanese cultivars of the common bean. Electrophoretically, the purified Tora-mame calmodulin migrated faster than bovine brain calmodulin in the presence or absence of Ca2+. The estimated molecular weight of Tora-mame calmodulin was 14,200 in the presenc of Ca2+ and 18,200 in the absence of Ca2+. Like other plant calmodulin, Tora-mame calmodulin lacked tryptophan and contained 1 mol each of tyrosine and half cystine per mol of protein. However, Tora-mame calmodulin has fewer acidic amino acid residues than other plant calmodulins did. Rat brain phosphodiesterase (Ca2+ –PDE) was stimulated by Tora-mame calmodulin, but the concentration of Tora-mame calmodulin, giving the half-maximal activation of Ca2+ –PDE, was higher than that of bovine calmodulin, and the maximal activity of Ca2+ –PDE obtained with Tora-mame calmodulin was lower than that obtained with bovine protein. The Ki value of W-7, a calmodulin a...

Hepatic calcium-binding protein regucalcin decreases Ca2+/calmodulin-dependent protein kinase activity in rat liver cytosol.

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on cytosolic Ca2+/calmodulin-dependent protein kinase activity was investigated. The increase in cytosolic Ca2+/calmodulin-dependent protein kinase activity with passage of incubation time was clearly prevented by the presence of regucalcin (1.0 microM). An appreciable effect of regucalcin was seen at 0.5 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (0.25-1.0 mM). This increase was clearly blocked by the presence of regucalcin (1.0 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2.5-15 micrograms). In the presence of regucalcin (1.0 microM), trifluoperazine (50 microM), an antagonist of calmodulin, significantly decreased the cytosolic Ca2+/calmodulin-dependent protein kinase activity. These results suggest that regucalcin can regulate the Ca2(+)-calmodulin effect in liver cytosol.