Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine from Lycium barbarum (Goji Berry) in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy.

Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB). We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression.

The plasma membrane calcium pump: a multiregulated transporter.

Activation of many cells, especially nonexcitable cells, results in a Ca(2+) transient that is influenced in part by the kinetics of active extrusion of Ca(2+) across the plasma membrane. The molecular cloning of the plasma membrane Ca(2+)-pump has helped to clarify the relationship between its structure and function. The Ca(2+)-pump is controlled by multiple regulators, including calmodulin, phospholipids and various kinases. Longer term control is achieved through regulation of its gene expression, and the presence of a number of Ca(2+)-pump isoforms that differ in their regulatory domains provides potential functional diversity. In this review, we focus on the mechanisms that regulate the function of the Ca(2+)-pump, and their physiological significance.

Pharmacodynamic interaction of warfarin with cranberry but not with garlic in healthy subjects.

BACKGROUND AND PURPOSE: Patients commonly take complementary medicines in conjunction with warfarin yet evidence supporting the safety or the risk of a herb-drug interaction is lacking. The aim of this study was to investigate the possible impact of two commonly used herbal medicines, garlic and cranberry, on the pharmacokinetics and pharmacodynamics of warfarin in healthy male subjects. EXPERIMENTAL APPROACH: An open-label, three-treatment, randomized crossover clinical trial was undertaken and involved 12 healthy male subjects of known CYP2C9 and VKORC1 genotype. A single dose of 25 mg warfarin was administered alone or after 2 weeks of pretreatment with either garlic or cranberry. Warfarin enantiomer concentrations, INR, platelet aggregation and clotting factor activity were measured to assess pharmacokinetic and pharmacodynamic interactions between warfarin and herbal medicines. KEY RESULTS: Cranberry significantly increased the area under the INR-time curve by 30% when administered with warfarin compared with treatment with warfarin alone. Cranberry did not alter S- or R-warfarin pharmacokinetics or plasma protein binding. Co-administration of garlic did not significantly alter warfarin pharmacokinetics or pharmacodynamics. Both herbal medicines showed some evidence of VKORC1 (not CYP2C9) genotype-dependent interactions with warfarin, which is worthy of further investigation. CONCLUSIONS AND IMPLICATIONS: Cranberry alters the pharmacodynamics of warfarin with the potential to increase its effects significantly. Co-administration of warfarin and cranberry requires careful monitoring.

Salacia oblonga root decreases cardiac hypertrophy in Zucker diabetic fatty rats: inhibition of cardiac expression of angiotensin II type 1 receptor.

AIMS: We investigated the effect of the water extract of Salacia oblonga (SOE), an ayurvedic antidiabetic and antiobesity medicine, on obesity and diabetes-associated cardiac hypertrophy and discuss the role of modulation of cardiac angiotensin II type 1 receptor (AT(1)) expression in the effect. METHODS: SOE (100 mg/kg) was given orally to male Zucker diabetic fatty (ZDF) rats for 7 weeks. At the end-point of the treatment, the hearts and left ventricles were weighed, cardiomyocyte cross-sectional areas were measured, and cardiac gene profiles were analysed. On the other hand, angiotensin II-stimulated embryonic rat heart-derived H9c2 cells and neonatal rat cardiac fibroblasts were pretreated with SOE and one of its prominent components mangiferin (MA), respectively. Atrial natriuretic peptide (ANP) mRNA expression and protein synthesis and [(3)H]thymidine incorporation were determined. RESULTS: SOE-treated ZDF rats showed less cardiac hypertrophy (decrease in weights of the hearts and left ventricles and reduced cardiomyocyte cross-sectional areas). SOE treatment suppressed cardiac overexpression of ANP, brain natriuretic peptide (BNP) and AT(1) mRNAs and AT(1) protein in ZDF rats. SOE (50-100 microg/ml) and MA (25 micromol) suppressed angiotensin II-induced ANP mRNA overexpression and protein synthesis in H9c2 cells. They also inhibited angiotensin II-stimulated [(3)H]thymidine incorporation by cardiac fibroblasts. CONCLUSIONS: Our findings demonstrate that SOE decreases cardiac hypertrophy in ZDF rats, at least in part by inhibiting cardiac AT(1) overexpression. These studies provide insights into a potential cardioprotective role of a traditional herb, which supports further clinical evaluation in obesity and diabetes-associated cardiac hypertrophy.

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells.

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression.

Involvement of calcium ions in the properties of cardiolipin-associated erythrocyte acetylcholinesterase.

Lipoprotein forms of acetylcholinesterase from bovine erythrocytes gave non-linear Arrhenius plots with a break at 20 degrees C and contained cardiolipin. The break in the Arrhenius plot was abolished by incubation of the enzyme in high salt (I = 1.8), but only in Ca2+ -chelating conditions. At I = 1.8 neither NaCl alone, CaCl2 nor sodium phosphate at acidic pH abolished the break. However, at this ionic strength either NaCl in 2 mM sodium phosphate (pH 7.4) or sodium phosphate, pH 8, or 1.0 M Na2CO3/NaHCO3 (pH 8.5--10, were able to remove the break. The Arrhenius plot break was regenerated by the addition of Ca2+ to the high salt-treated enzyme with mild homogenization, but could not be regenerated in the presence of EDTA unless CaCl2 was added in excess of the EDTA. Conditions which abolished the break enabled endogenous cardiolipin to be removed from the enzyme by chloroform/methanol extraction Cardiolipin from acetylcholinesterase incubated in high salt in Ca2+ -chelating conditions was not accessible to digestion by phospholipase A2, and was not separated from the enzyme by flotation in a sucrose density gradient or by Sephadex G-200 chromatography. Thus both Ca2+ and cardiolipin appear to be inaccessible, possibly by being tightly associated in the hydrophobic core of the enzyme by ionic and hydrophobic forces. Ca2+ may modulate the temperature dependence of acetylcholinesterase activity through a functionally linked ionic interaction with the enzyme-cardiolipin complex.

Influence of EGTA on the apparent Ca2+ affinity of Mg2+-dependent, Ca2+-stimulated ATPase in the human erythrocyte membrane.

The apparent Ca2+ affinity of Mg2+-dependent, Ca2+-stimulated ATPase (Mg2+ + Ca2+)-ATPase) in human erythrocyte membranes increased with increasing concentrations of EGTA used to buffer free Ca2+. The shift in apparent Ca2+ affinity was seen in membranes prepared by hypotonic hemolysis and in membranes depleted of endogenous activators by EDTA treatment. The effect of EGTA differed from that of calmodulin, as it increased Ca2+ affinity without increasing V. EGTA also increased the apparent Ca2+ affinity when calmodulin was present in the assay medium. ATP-stimulated calcium binding to membranes was greater at 1 mM EGTA than at 0.1 mM EGTA. Similarly to ATPase activation, whereas binding decreased as Ca2+ was raised above 35 microM at 1.0 mM EGTA, binding progressively increased up to 100 microM or more free Ca2+ at 0.1 mM EGTA. EGTA also increased the Ca2+ affinity of Triton X-100-solubilized (Mg2+ + Ca2+)-ATPase, indicating that its effect did not depend on an intact membrane. Analysis of the kinetic data by a computerized nonlinear curve fitting procedure showed that a low Ca2+ affinity state of the enzyme was converted to a high Ca2+ affinity state in the presence of EGTA. The species associated with the enzyme interconversion appeared to be [CaEGTA]2-.

Calcium-dependent inhibition of the erythrocyte Ca2+ translocating ATPase by carbodiimides.

The ATP hydrolytic activity of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte plasma membrane was strongly inhibited by the nonpolar compound, N,N'-dicyclohexylcarbodiimide, both in the presence and in the absence of calmodulin. However, the more water-soluble carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide had little inhibitory effect on the enzyme. The inhibitory effect of N,N'-dicyclohexylcarbodiimide was most pronounced at acid pH, and declined sharply at alkaline pH values. In addition, the optimum pH for the enzyme activity also shifted to more alkaline values in the presence of the carbodiimide. Calcium ion appears to favor the inhibition induced by the carbodiimide, in contrast to the observed protection by Ca2+ in the sarcoplasmic reticulum Ca2+-translocating ATPase. N,N'-Dicyclohexylcarbodiimide also dramatically decreased the stimulatory effect of calmodulin on the activity of the enzyme.

Differential behaviour of eel and erythrocyte acetylcholinesterase on N-methylacridine affinity columns. Importance of ligand affinity and concentration.

The retention and elution of acetylcholinesterase from bovine erythrocytes and electric eel on N-methylacridinium affinity columns have been compared at various ligand concentrations. A soluble 7.7 S dimeric form of bovine erythrocyte acetylcholinesterase required a ligand concentration of 2.0-2.8 mumol/ml in 0.1 M NaCl for retention, compared to 0.44 mumol/ml for various forms of the electric eel acetylcholinesterase. The difference in the retention of acetylcholinesterase from these two sources could not be explained by differences in their oligomeric structure. The affinity of bovine erythrocyte acetylcholinesterase for N-methylacridinium was 13-fold or more lower than the electric eel acetylcholinesterase at similar ionic strengths. N-Methylacridinium appeared to react selectively with the catalytic anionic site of both enzymes. It was concluded that the affinity of the side arm ligand was the major determinant of the differences in the retention properties of the eel and erythrocyte acetylcholinesterase. The difference in affinity for N-methylacridinium probably reflects differences in the organic cation binding region of the two enzymes.

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane.

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.

The effect of calmodulin on the interaction of carbodiimides with the purified human erythrocyte (Ca2+ + Mg2+)-ATPase.

The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel.

Membrane-bound high molecular mass proteinases from human erythrocytes.

Two high molecular mass proteinases, multicatalytic proteinase (MCP) and a new high molecular mass proteinase (HMP) with only chymotrypsin-like activity (Khan et al. (1994) J. Biol. Chem. 269, 10016-10021) from human erythrocyte membranes, have been compared. For this purpose, MCP was purified from human erythrocyte membranes in the active form towards synthetic peptide substrates; it also hydrolysed the protein substrates [14]methyl casein and [14C]oxidised insulin beta chain at 37 degrees C. MCP from plasma membranes exhibited hollow cylindrical structures also typical of cytosolic forms. Radiolabelled diisopropyl fluorophosphate, [3H]DFP, a serine proteinase inhibitor, labelled a band of Mr 23 000 in membrane MCP. By contrast, no labelling was obtained with HMP. Chymotrypsin-like activity of HMP was also found to be insensitive to DFP. On the other hand, DFP inhibited chymotrypsin-like and peptidylglutamyl peptide hydrolysing activities of membrane MCP, with no effect on its trypsin-like activity. The inhibition of MCP by DFP was concentration-dependent. These studies showed that MCP and HMP represent two distinct kinds of proteinases with chymotrypsin-like activities and can be distinguished by the serine proteinase inhibitor DFP.

The effect of thrombin and serine proteases on intracellular Ca2+ in rat aortic smooth muscle cells.

Cultures of vascular smooth muscle cells (VSMC) are commonly used to study the events and defects found in hypertension and atherosclerosis. In particular Ca2+ homeostasis in cellular signalling has been the focus of extensive research. Since trypsin has been shown to mobilise Ca2+ in some cell types, we have investigated its effect on various aspects of Ca2+ homeostasis in rat aortic smooth muscle cells (RASMC). The effects of trypsin, alpha-chymotrypsin and elastase (other serine proteases) on intracellular Ca2+ in cultured aortic cells isolated from Wistar rats have been investigated. Trypsin (24 micrograms/ml) elicits intracellular Ca2+ mobilisation, after which cells become nonresponsive to thrombin Ca2+ mobilisation but retain responsiveness to Angiotensin II (AII). alpha-Chymotrypsin (24 micrograms/m) inhibits the thrombin Ca2+ mobilising response, without itself initiating a Ca2+ transient or affecting AII Ca2+ mobilisation. Elastase (24 micrograms/ml) was not effective in mobilising intracellular Ca2+ or inhibiting the thrombin response. We have also observed diminished thrombin Ca2+ mobilisation responses between cells in suspension and cell monolayers, which appeared to be unrelated to proteolysis but due to morphological changes of the cells. Our results suggest that trypsin acts on the thrombin receptor via a specific proteolysis mechanism to mobilise intracellular Ca2+ ([Ca2+]i) in RASMC. The amount of Ca2+ released by thrombin or trypsin is dependent on the morphology of the cell and the state of the tethered ligand of the thrombin receptor exposed by the protease.

Ca2+ and calmodulin-sensitive inositol trisphosphate kinase from bovine parathyroid.

A Ca2+ and calmodulin-activated inositol 1,4,5 trisphosphate kinase activity was detected in both soluble and membrane fractions from bovine parathyroid glands. Ca2+ activated the soluble enzyme in the concentration range 100 nM to 1 microM, which corresponds to the Ca2+ concentration range observed in the intact cell following maximal variation in extracellular Ca2+, the principal regulator of parathyroid hormone release. The Ca2+ sensitivity of the enzyme was absolutely dependent upon calmodulin. A similar activity was detected in the membranes but could be progressively removed by repeated washing at low ionic strength. This, together with data demonstrating binding of the enzyme to the hydrophobic matrix, Phenyl-Sepharose, suggests that the association of the enzyme with the membrane is likely to involve a significant hydrophobic component. The organic base, amiloride was identified as an inhibitor of the activity, the degree of inhibition being most marked in the presence of Ca2+ and calmodulin (K0.5 approx. 0.1 mM). The Ca2+ concentration dependence of the IP3 kinase suggests that inositol 1,3,4,5 tetrakisphosphate may be a messenger in the signal transduction pathway for the feedback inhibition of PTH secretion by extracellular Ca2+.

The plasma membrane calcium pump--a physiological perspective on its regulation.

This review focuses on the physiological role of the plasma membrane Ca(2+)+ Mg(2+)-dependent adenosine triphosphatase (PM Ca(2+)-ATPase) in cellular signalling. Particular attention has been paid to the regulation of the PM Ca(2+)-ATPase (PM Ca2+ pump) by calmodulin, proteases, protein kinases, acidic phospholipids and oligomerization in intact cells. We also review recent work investigating the possible regulation of the PM Ca2+ pump by G proteins and agonists. The source of adenosine triphosphate (ATP) and Ca2+ in fueling and activating the Ca2+ pump is discussed, as well as the possible role of the PM Ca(2+)-ATPase in subplasma membrane Ca2+ regulation. The physiological implication of the localisation of the PM Ca2+ pump in caveolae is also considered.

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin.

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum.

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.

Antagonism of calmodulin and phosphodiesterase by nifedipine and related calcium entry blockers.

Nifedipine, a 1,4-dihydropyridine Ca2+ entry blocker, partially inhibits calmodulin-activated and, to a lesser extent, basal (non-activated) cyclic AMP phosphodiesterase activity at 10-440 microM. The inhibition of calmodulin-activated phosphodieserase does not parallel Ca2+ entry blockade, since analogs of nifedipine, which are 500-fold less potent than nifedipine as Ca2+ entry blockers (Bolger et al. (1982) Biochemical and Biophysical Research Communications 104, 1604-1609), are equal in potency to nifedipine as calmodulin-activated phosphodiesterase inhibitors. Furthermore, the inhibition of calmodulin-activated phosphodiesterase by nifedipine is about 500-fold less potent than its inhibition of Ca2+ entry blockade. It is suggested that the low affinity interaction of nifedipine and related 1,4-dihydropyridines with calmodulin and phosphodiesterase is also of low specificity and therefore is unlikely to contribute to the cardiac and vascular muscle relaxant actions of these drugs at normal pharmacological concentrations.

Investigation of (Ca2+ + Mg2+)-ATPase phosphoprotein formation in erythrocyte membranes of patients with cystic fibrosis.

The (Ca2+ + Mg2+)-ATPase present per mg of protein in erythrocyte membranes of controls and patients with cystic fibrosis (CF) was determined by estimation of the levels of its phosphoprotein. In the presence of 10 mM free Ca2+, which inhibits phosphoprotein decomposition, significantly less phosphoprotein intermediate, ECaP, was found in erythrocyte membranes from CF patients than in age- and sex-matched controls; this correlated with a significant decrease in (Ca2+ + Mg2+)-ATPase activity. These observations indicate a decrease in the number of functional (Ca2+ + Mg2+)-ATPase molecules in erythrocyte membranes from CF patients or an alteration in either the structure of the pump protein or the composition of its environment.

Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes.

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.