Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Studies on the kinetics of cation-associated fluorescence changes in chloroplast membranes

A soluble Ca²⁺- and Ca²⁺—calmodulin-activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca²⁺-dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3-fold and up to 16-fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca²⁺-dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca²⁺-dependent fashion. This type of Ca²⁺-activated protein kinase may be involved in stimulus—response coupling in plants.     

The role of calmodulin on Ca2+-dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca²⁺-induced loss of KCl from human red cells. However, the K_i values for this effect did not bear good correlation with the K_i values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KCl fluxes were also affected. Added calmodulin did not have any effect on Ca²⁺-dependent ⁸⁶Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca²⁺-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on ⁸⁶Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations, with or without modification of the relative rate of ⁸⁶Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations without affecting the relative rate of ⁸⁶Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported K_i for calmodulin-dependent systems. These results suggest that Ca²⁺-dependent K⁺ channels of red cells are not regulated by calmodulin.     

Tetrahymena calmodulin. Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in Tetrahymena

A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlony's immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium, but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti-Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.     

EGTA induces the synthesis in Escherichia coli of three proteins that cross-react with calmodulin antibodies

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca²⁺ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca²⁺ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca²⁺-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca²⁺ ionophore A23187 + Ca²⁺, may be defective in the regulation of the level of free intracellular Ca²⁺.     

Calcium- and calmodulin-regulated phosphorylation of soluble and membrane proteins from corn coleoptiles

In vitro phosphorylation of several membrane polypeptides and soluble polypeptides from corn (Zea mays var. Patriot) coleoptiles was promoted by adding Ca²⁺. Ca²⁺-promoted phosphorylation of the membrane polypeptides was further increased in the presence of calmodulin. Both Ca²⁺-stimulated and Ca²⁺- and calmodulin-stimulated phosphorylations of membrane polypeptides were inhibited by chlorpromazine, a calmodulin antagonist. Ca²⁺-stimulated phosphorylation of soluble polypeptides increased with increasing Ca²⁺ concentration. The calmodulin antagonists chlorpromazine and trifluoperazine inhibited the Ca²⁺-promoted phosphorylation of soluble polypeptides. Added calmodulin promoted the Ca²⁺-dependent phosphorylation of a 98 kilodaltons polypeptide. Both Ca²⁺-dependent and Ca²⁺-independent phosphorylations required Mg²⁺ at an optimal concentration of 5 to 10 millimolar. Cyclic AMP was found to have no stimulatory effect on protein phosphorylation. Sodium molybdate, an inhibitor of protein phosphatase, increased the net phosphorylation of several polypeptides. Rapid loss of radioactivity from the phosphorylated polypeptides following incubation in unlabeled ATP indicated the presence of phosphoprotein phosphatase activity.     

20-Hydroxyecdysone control of the synthesis of putative calcium-binding proteins in the epidermis of Tenebrio molitor

The altered pattern of synthesis of putative calcium-binding proteins (pCaBPs) in the mid-instar epidermis following exposure to 20-hydroxyecdysone (20HE) in vitro was followed through the incorporation of [³⁵S]methionine into newly synthesized polypeptides. pCaBPs were separated from other epidermal polypeptides by Ca²⁺-dependent hydrophobic interaction chromatography, followed by polyacrylamide gel electrophoresis and fluorography. The dominant effect of 20HE is to depress pCaBP synthesis. Of the 17 newly-synthesized pCaBPs consistently detected in total cell lysates, the synthesis of ten was depressed strongly and that of the remaining seven was unaltered by exposure to 20HE. Most newly-synthesized pCaBPs identified were found in the cytosolic fraction of the epidermis. One pCaBP was identified as Tenebrio calmodulin based on its altered electrophoretic mobility in the absence of calcium ions, its isoelectric focusing point, its binding to phenyl-Sepharose and phenothiazine and its binding to antibodies against purified mammalian calmodulin. The synthetic rate of this pCaBP was not affected by 20HE. The distribution of another pCaBP (32 kDa) shifted from the cytosolic to the microsomal fraction on adding Ca²⁺ (or reversed by adding EGTA) to the cell extract before fractionation. The synthesis of this protein was depressed by 20HE. These findings suggest that 20HE influences epidermal behaviour, at least in part, through its ability to modulate the synthetic rate of several pCaBPs.     

Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae

Ca²⁺-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca²⁺- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species. At 0.2 μM beef calmodulin the phosphorylation was optimal at 3 μM free Ca²⁺, and at 80 μM free Ca²⁺ it was half-maximal at about 0.1 μM beef calmodulin. It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca²⁺-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca²⁺ (K_0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca²⁺-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with ³²P-phosphate. Overall, the results show the existence of a Ca²⁺-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca²⁺-dependent phosphorylation.     

Goat testis calmodulin: Purification and physicochemical characterization

Calmodulin has been purified in large quantities from goat (Capra hiscus) testis. The procedure includes heat treatment, hydrophobic interaction chromatography, and gel filtration. Goat testis calmodulin closely resembles other mammalian testis calmodulin studied so far. The protein has an extinction coefficient value (E _1cm ^1% ) of 2.09 at 280 nm, a Stokes radius of 23.2 Å at 0.15 M KCl, and a frictional ratio of 1.38. Ca²⁺, and Tb³⁺ binding studies demonstrate that the protein has four Ca²⁺-binding sites with aK _d of 52.5 µM. Goat testis calmodulin shows close similarity to other calmodulins in the amino acid composition and in demonstrating an altered migration on SDS/PAGE upon Ca²⁺ binding. The protein also exhibits anomalously high values for molecular weight and Stokes radius as determined from the analytical gel chromatography and a change in its elution volume with the change of salt concentration in the eluant. These results have been discussed in view of the recently available knowledge from the crystallographic studies of rat testis calmodulin.     

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca²⁺-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca²⁺-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca²⁺-binding loops. We examined Ca²⁺ binding of the protein by a Ca²⁺-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca²⁺-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Properties of calcium-dependent regulatory proteins from fungi and yeast

Calmodulins were isolated from vegetative mycelia of Basidiomycetes fungi, Agaricus campestris and Coprinus lagopus. These calmodulins showed similar mobilities to those of animal calmodulins on nondenaturing polyacrylamide gel electrophoresis in the presence or absence of Ca2+. The molecular weights of both calmodulins were determined to be 16,000. Agaricus calmodulin consisted of 148 amino acids including epsilon-N-trimethyllysine and cysteine. The UV-absorption spectrum showed the relatively high content of phenylalanine in Basidiomycetes calmodulins. The difference UV-absorption spectrum due to the blue shift by Ca2+ was observed. Both calmodulins activated muscle myosin light chain kinase and pea NAD+ kinase in a Ca2+-dependent manner, and the activities were inhibited by trifluoperazine or chlorpromazine. A calmodulin-like protein was partially purified from baker's yeast, Saccharomyces cerevisiae. However, detection of a calmodulin-like protein in prokaryotes was not successful.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.