Autolysis of phospholipids in homogenates of various plant tissues

The rates of autolysis of endogenous phospholipids were measured in homogenates of 17 cultivated plant tissues. For seven out of the 17 samples, the rates of autolysis of phosphatidylcholine (PC) were greater than 10% hydrolysis per hr (at pH 7.5 and 4°). The addition of dibucaine (2 mM) inhibited the autolytic rates in homogenates of 12 out of 17 samples. The only plant homogenates where dibucaine stimulated the autolytic rates were those of potato tubers. Although dibucaine inhibited the rate of autolysis of PC in potato leaf homogenates there was no advantage to adding it during homogenization and preparation of differential centrifugation fractions from potato leaves. Homogenization with different types and concentrations of osmotica affected the rates of autolysis of PC. Buffering the homogenates at pH 8 drastically inhibited the rates of autolysis in potato leaf homogenates but had little effect on bean leaf homogenates. Various strategies for controlling the rate of membrane lipid breakdown in different plant extracts are discussed.     

Autolysis of membrane lipids in potato leaf homogenates: Effects of calmodulin and calmodulin antagonists

Potato leaves contain high levels of lipolytic acyl hydrolase activity which degrades phospholipids and galactolipids during homogenization and organelle isolation. Four calmodulin antagonists (dibucaine, tetracaine, trifluoperazine a and chlorpromazine) were found to inhibit the rate of hydrolysis of endogenous membrane lipids in homogenates of potato leaves. In contrast, the addition of calcium and purified calmodulin stimulated the rate of hydrolysis. These results indicate that a lipolytic acyl hydrolase activity in potato leaves appears to be mediated either directly or indirectly by calcium and calmodulin.     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation,H+-ATPase activity,calmodulin-dependent cyclic nucleotide phosphodiesterase activity,and calmodulin binding

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited ∼40% by trifluoperazine or tamoxifen with half-maximal effects at 1–3 μM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at ∼100 nM, cischroman and CDRI 85/287 gave 15–20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 μM. However, ATP-dependent membrane vesicle acridine transport showed that H⁺-ATPase activity was abolished by all compounds with 50% effects at 0.25–1 μM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H⁺-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells. J. Cell. Biochem. 66:358–369, 1997. © 1997 Wiley-Liss, Inc.     

Calmodulin is not involved in the regulation of exogenous NADH oxidation by plant mitochondria

The oxidation of exogenous NADH by mitochondria from potato ( Solanum tuberosum L., cv. Bintje) tubers, measured with different electron acceptors, oxygen, cytochrome c , duroquinone and ubiquinone 1, was greatly enhanced under high salt conditions compared to low salt conditions, confirming the stimulatory effect of electrostatic screeening of negative membrane charges by cations. In addition to this nonspecific stimulation, the oxidation of exogenous NADH showed a specific dependence on Ca²⁺. Results presented here suggest that calmodulin was not directly involved in the regulation of exogenous NADH oxidation by potato mitochondria: (1) Calmodulin antagonists were found to inhibit electron flow at several sites in a nonspecific manner. (2) Using a phenothiazine-Affi Gel column, it was not possible to demonstrate the presence of calmodulin in Triton X-100 solubilized mitochondria. (3) Fractions eluted from a calmodulin-Sepharose column with EGTA [ethyleneglycolbis (β-aminoethylether)-N, N, N', N'-tetraacetic acid] did not display any activity related to mitochondrial electron transport, suggesting that NADH dehydrogenase had no specific affinity for calmodulin. The possible indirect involvement of calmodulin in the regulation of exogenous NADH oxidation by Ca²⁺ is discussed.     

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca²⁺/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca²⁺ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca²⁺ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca²⁺/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Calmodulin is involved in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary chromaffin cells.

The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal medullary chromaffin cells. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. Unlike calmodulin, addition of caldesmon, actin or bovine serum albumin did not increase the release. Calmodulin increased the release at Ca2+ concentrations of more than 10(-6) M and its effect increased with increase in Mg2+ concentration. Th release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin, which specifically inhibits exocytotic secretion. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells.     

Calmodulin antagonists inhibit Ca2+ uptake of mitochondria of guinea pig peritoneal macrophages

The Ca²⁺ uptake of the mitochondria of guinea pig peritoneal macrophages was not stimulated by the addition of calmodulin. However, calmodulin antagonists, both phenotiazines and N-naphthalenesulfonamides, in low concentrations inhibited the Ca²⁺ uptake of the mitochondoria, as compared to the inhibition of the calmodulin-dependent stimulation of brain phosphodiesterase. These calmodulin antagonists appear to have severe side effects on active processes of the mitochondria and which are unrelated to the specific effect on calmodulin.     

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins.

Calmodulin was purified from human tonsillar lymphocytes utilizing calcium-dependent binding of calmodulin to fluphenazine-Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H-thymidine incorporation into DNA of lectin-stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H-thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca(2+)-calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.     

Peptide analogs of the pseudosubstrate domain of smooth muscle myosin light chain kinase inhibit actomyosin ATPase activity at concentrations that do not inhibit superprecipitation.

The inhibitory effect of calmodulin antagonists, synthetic peptide analogs of the pseudosubstrate domain of smooth muscle MLC kinase, and an inhibitor based on the sequence of MLC were examined using bovine aortic actomyosin and isolated chicken gizzard MLC. Much lower concentrations of the peptides were necessary to inhibit actomyosin ATPase activity than to inhibit superprecipitation. In contrast, calmodulin antagonists inhibited both ATPase activity and superprecipitation at similar concentrations. The peptide analogs were competitive with isolated MLC, but not calmodulin, for inhibition of MLC kinase. These results suggest that in addition to the calmodulin dependence of MLC phosphorylation, a second calmodulin-like protein may be important in actin-myosin interactions. The data also suggest that the pseudosubstrate hypothesis may not completely account for regulation of MLC kinase activity.     

Ca2+ -dependent protein phosphorylation in germinated pollen of Nicotiana alata, an ornamental tobacco

The 40 000 g supernatant and 40 000 g pellet from extracts of germinated pollen of Nicotiana alata Link et Otto contain protein kinase activity which catalyzes the phosphorylation of histones, casein and a range of endogenous polypeptides. Phosphorylation of certain low-molecular-weight, casein-derived polypeptides is activated at low (12–37 μ M ) and partially inhibited at higher (540 μ M ) concentrations of free Ca²⁺. Histone phosphorylation is largely Ca²⁺-dependent and is activated by 540 μM free Ca²⁺. No activation of protein phosphorylation by micromolar concentrations of calmodulin is found, but phenothiazine-derived calmodulin antagonists markedly stimulate protein phosphorylation.     

Possible role of calmodulin in the secretion of Entamoeba histolytica electron-dense granules containing collagenase

Entamoeba histolytica cells secrete electron-dense granules (EDGs) that have collagenase activity. To study the possible involvement of calmodulin (CaM) on EDG secretion, the effect of several CaM antagonists (TFP, R24571, W-7, W-5, dibucaine and DL-propranolol) was tested on this cellular function. Except for W-5 and dibucaine, the rest of these compounds inhibited EDG secretion. Transmission electron microscopy of collagen-activated trophozoites showed numerous EDGs located in or near the surface membrane. In contrast, trophozoites incubated with TFP showed no EDGs. Protein kinase C inhibitors (H-7, ML-9) had no effect on EDG secretion, suggesting that CaM antagonists acted by selectively inhibiting CaM. These results suggest that a CaM-dependent process is involved in EDG secretion.     

Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane

The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.     

Membrane-degrading enzymes in the leaves of Solanum tuberosum

When homogenates of potato leaves were prepared under conditions which are typical for organelle isolation (pH 7.5 and 4°), membrane lipids underwent rapid hydrolysis (17% of phosphatidylcholine was hydrolysed in 2 hr). Leaves of 41 potato cultivars were surveyed for phospholipase activity to determine whether certain cultivars might be more suitable for the preparation of organelles. Phospholipase activities ranged from 1.04 to 11.60, μmol/min · g fr. wt and p-nitrophenyl palmitate hydrolase activity ranged from 0.0119 to 0.0502,μmol/min · g fr. wt. These phospholipase values were several hundred-fold higher than previously reported for potato leaves and nearly as high as in potato tubers. Most of the phospholipase activity in leaves was soluble and not membrane-associated as previously reported.     

Phospholipid base exchange in human leukocyte membranes: quantitation and correlation with other phospholipid biosynthetic pathways

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca2+ concentration, but markedly inhibited by Mg2+. Despite this Ca2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05-0.2 mg/ml; Con A, 0.5-2 micrograms/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N-transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A2 activity.     

Metabolism of lysophosphatidyl ethanolamine and lysophosphatidyl choline by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages

A comparison has been made between the conversion of (32)P-labeled lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl choline (LPC) to their respective acylated and deacylated derivatives by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages. Synthesis of PE by both homogenates and of PC by macrophage homogenates proceeded to about the same extent and is attributed to direct acylation of the lyso compounds. At higher LPC concentrations formation of PC by leukocytes is far greater than by macrophages. The mechanism of this enhanced synthesis of PC, which is brought out by higher substrate concentrations, is believed to be a transfer of the acyl group of one LPC molecule to another. Under optimal conditions macrophage homogenates deacylated LPE to a greater extent than LPC, while the reverse was true for leukocyte homogenates. Albumin inhibited deacylation of LPC and its conversion to PC by leukocytes, perhaps by binding the substrate (2 moles of LPC per mole of albumin). Other effects of albumin-stimulation of deacylation and acylation of LPE by macrophages, inhibition of deacylation and acylation of LPE by leukocytes-remain unexplained.     

Noncompetitive, Ca(2+)-independent inhibition of pyruvate dehydrogenase phosphatase by fluphenazine.

The effects of two different classes of calmodulin antagonists on the catalytic activities of purified pyruvate dehydrogenase (PDH) phosphatase and PDH complex (PDC) were studied. In general, PDH phosphatase was more strongly inhibited than PDC by the calmodulin antagonists with the following potency order: fluphenazine > chlorpromazine > thioridazine > triflupromazine. Promazine and two sulfonamides (W-5 and W-7) did not suppress PDH phosphatase activity at 1 mM concentrations, while about 20% of PDC activity was inhibited by these antagonists. Fluphenazine-mediated inhibition of PDH phosphatase was observed with the purified PDC as well as intact mitochondria. Although Ca2+ stimulates PDH phosphatase activity, the addition of exogenous Ca2+ did not overcome the inhibition by calmodulin antagonists. These results suggest that the suppression of PDH phosphatase activity is dependent upon the structure of the individual calmodulin antagonist and appears to be Ca(2+)-independent. Kinetic analysis showed a noncompetitive inhibition of PDH phosphatase by fluphenazine, indicating that it binds to different site(s) from the catalytic site of the enzyme.     

Enhancement of chemiluminescence of the neutrophils by low concentrations of trifluoperazine

One to 10 μM trifluoperazine was found to potentiate luminol-dependent chemiluminescence of neutrophils induced by n-formyl-methionyl-leucylphenyalanine. It did not potentiate chemiluminescence induced by A23187 or by phobor myristate acetate. Low concentrations of another calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, an intracellular Ca⁺⁺ antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, and a local anesthetic dibucaine, were found to possess similar activity. It is suggested that trifluoperazine potentiates chemiluminescence by acting on certain cellular processes that follow after stimulation by n-formyl-methionyl-leucylphenylalanine, but not by A23187 or by phorbor myristate acetate, and that this effect may be calmodulin-independent.     

Ca(II)-calmodulin regulation of morphological commitment in Ceratocystis ulmi

Abstract The commitment phenomenon exhibited by Ceratocystis ulmi is a manifestation of the requirement for a Ca(II)-calmodulin interaction for mycelial growth. Under otherwise identical conditions, addition of CaCl₂(10 mM) to committed yeasts caused them to germinate, while addition of the calmodulin antagonists chlorpromazine (80 μM), dibucaine (750 μM), or trifluoperazine (60 μM) to committed mycelia caused them to bud.