Polymyxin B is a more selective inhibitor for phospholipid-sensitive Ca2+-dependent protein kinase than for calmodulin-sensitive Ca2+-dependent protein kinase

Polymyxin B inhibited phospholipid-sensitive Ca²⁺-dependent protein kinase competitively with respect to phosphatidylserine (a phospholipid cofactor), with a K_i of 1.8 μM. It also inhibited myosin light chain kinase (a calmodulin-sensitive species of Ca²⁺-dependent protein kinase) competitively with respect to calmodulin, but with a higher K_i of 17.0 μM. Bacitracin, another polypeptide antibiotic, was much less active in inhibiting both enzymes. Polymyxin B and bacitracin were without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The findings indicate that polymyxin B, a surface active agent, effectively inhibited the phospholipid-sensitive enzyme presumably by interacting with phosphatidylserine.     

Ca2+-dependent cross-linking processes in human platelets

When platelet cytoplasmic Ca²⁺ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking. [¹⁴C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIB and III, actin and tropomyosin. The lose of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca²⁺-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca²⁺-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca²⁺-dependent protein kinase from pig testis. Gossypol inhibited Ca²⁺-dependent activity of the enzyme without affecting its basal activity. The IC₅₀ value (concentration causing 50% inhibition) was 31 μM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (K_i = 31 μM) or lysine-rich histone (K_i = 30μM), and competitively with respect to phosphatidylserine (K_i = 2.1 μM). With Ca²⁺, irrespective of the presence or absence of 1,3-diolein, the compound lowered V_max and increased the apparent K_a for Ca²⁺. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrate in the testis for the enzyme located in nucleosome), with an IC₅₀ value of 88 μM. These results suggested that a phospholipid-sensitive Ca²⁺-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Comparative abilities of lanthanide ions La3+ and Tb3+ to substitute for Ca2+ in regulating phospholipid-sensitive Ca2+-dependent protein kinase and myosin light chain kinase

Although lanthanide ions La³⁺ and Tb³⁺ were only slightly able to substitute for Ca²⁺ to activate phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), they potentiated the ability of a suboptimal concentration of Ca²⁺ to stimulate the enzyme. In comparison, the lanthanides were much more effective Ca²⁺ substitutes for myosin light chain kinase, a calmodulin-sensitive Ca²⁺-dependent protein kinase. Both enzymes, however, were inhibited by high concentrations of lanthanides either in the presence or absence of Ca²⁺. Similar effects of the lanthanides were also noted on phosphorylation of endogeneous substrates in the particulate fraction of rat brain stimulated by either phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The La³⁺- or Tb³⁺-stimulated activity of PL-Ca-PK, as the Ca²⁺-stimulated activity, was inhibited by various agents, such as trifluoperazine, polymyxin B, cobra cytotoxin I, melittin, and spermine.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Ionophore monensin induces Na+-dependent secretion from rabbit neutrophils. Requirement for intracellular Ca2+ stores

Rabbit (and human) neutrophils release the secretory enzyme β-glucuronidase when treated with the ionophore monensin in the presence of Na⁺. Release of β-glucuronidase occurs without loss of the cytosol enzyme lactate dehydrogenase and a number of other features of the release process lead us to conclude that a normal exocytotic mechanism is involved. These include sensitivity to metabolic inhibition, enhancement of release induced by cytochalasin B and a requirement for internal sources of Ca²⁺ when the cells are stimulated with monensin in the absence of extracellular Ca²⁺. The release process due to monensin differs from that due to receptor directed agonists such as fMet-Leu-Phe and the Ca²⁺ ionophores A23187 and ionomycin in respect of a prolonged time-course which extends over 20 min; nor do monensin-stimulated neutrophils generate the superoxide anion. The results are discussed in the light of reports which indicate a rôle for Na⁺ in the activation of neutrophils by other ligands.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca²⁺-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A₂ (cPLA₂ε) was recently identified as a Ca²⁺-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA₂ε function as Ca-NAT. We next purified both mouse recombinant cPLA₂ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca²⁺ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1?mM CaCl₂ and lowered the EC₅₀ value of Ca²⁺ >8-fold. Using a PS probe, we showed that cPLA₂ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA₂ε with PS in living cells. Finally, we found that the Ca²⁺-ionophore ionomycin increased [¹⁴C]NAPE levels >10-fold in [¹⁴C]ethanolamine-labeled cPLA₂ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca²⁺-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca²⁺-dependent activity and human cPLA₂ε isoforms also functioned as Ca-NAT.     

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540–3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of cadmodulin did not require Ca²⁺ for activation of the enzyme; and activation occured in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca²⁺. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased V_max and with no changes in the F_m (ATP). High levels of cation, up to 100 mM Mg²⁺, did not effect the action of cadmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.     

Determination of Ca2+- and phospholipid-dependent protein kinase in rat liver membranes

A method has been developed to measure the Ca²⁺- and phospholipid-dependent protein kinase in membrane fractions. The method is based on the fact that this enzyme is resistant to comparatively high concentrations of octyglycoside. Rat liver membranes were treated with octylglycoside and the phosphate incorporation from |-³²P]ATP was measured in the presence of histone H1. The enzyme activity was determined as the difference between the incorporation obtained after addition of Ca²⁺ and phosphatidylserine and the incorporation obtained without these additions but with EGTA. The endogenous incorporation of phosphate to membrane components was constant under these incubation conditions. The conditions for determination of the membrane-bound enzyme were optimized. Two thirds of the total enzymic activity was attached to membranes in rat liver cells. A highly purified plasma membrane preparation had the highest specific activity, while most of the bound enzyme was found in microsomes, an only traces were found in mitochondria.     

The Ca2+-dependent DNases are Involved in Secondary Xylem Development in Eucommia ulmoides

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca 2+ -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca 2+ -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca 2+ -dependent DNases are involved in secondary xylem development.     

ATP and Ca2+ binding by the Ca2+ pump protein of sarcoplasmic reticulum

The binding of ATP and Ca²⁺ by the Ca²⁺ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca²⁺ pump protein has been found to contain one specific ATP and two specific Ca²⁺ binding sites per phosphorylation site. ATP binding is dependent on Mg²⁺ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca²⁺. In the presence of Mg²⁺ and the absence of Ca²⁺, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca²⁺ binding. In the absence of ATP, Ca²⁺ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg²⁺, Sr²⁺ and La³⁺, in that order, decrease Ca²⁺ binding by the Ca²⁺ pump protein. The affinity of the Ca²⁺ pump protein for both ATP and Ca²⁺ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca²⁺ pump protein-MgATP^2? and Ca²⁺ pump protein-calcium complexes are approx. 0.25 and 0.5 μM^?1, respectively. The unphosphorylated Ca²⁺ pump protein does not contain a Mg²⁺ binding site with an affinity comparable to those of the ATP and Ca²⁺ binding sites.The affinity of the Ca²⁺ pump protein for Ca²⁺ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca²⁺ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca²⁺. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca²⁺.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca₂²⁺-MgATP^2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca²⁺ and probably Mg²⁺.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

The peripheral Ca2+ pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca²⁺ or Mg²⁺ and was not affected by F-actin and ouabain. V_max was 2.8 and 10.0 μmol P_i/mg protein per h for Ca²⁺- and Mg²⁺-dependent activity, respectively, and the corresponding K_m was 4.8·10^?4 M and 3.2·10^?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca²⁺-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca²⁺-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca²⁺ for activation.     

Characterization of Ca2+-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.