Basil D. Roufogalis

Phenothiazine antagonism of calmodulin: A structurally-nonspecific interaction

Abstract Phenothiazine antagonism of calmodulin-stimulated (Mg 2+ + Ca 2+ )-ATPase activity in erythrocyte ghosts was examined to determine the structural specificity of the interaction. Four analogs of chlorpromazine, differing in the position of the chlorine substitution of the aromatic ring, were about equally potent in antagonizing calmodulin activation, while only the 2-chloro analog (chlorpromazine) has tranquilizer activity and antagonizes dopamine-sensitive adenylate cyclase. As all four analogs have similar hydrophobicity and surface activity, the results indicate that the antagonism of calmodulin by phenothiazines is unrelated to their pharmacological specificity and occurs at a structurally-nonspecific hydrophobic site on the protein.

Determination of the stoichiometry of the calcium pump in human erythrocytes using lanthanum as a selective inhibitor.

Since the discovery of ATP dependent calcium transport in human erythrocyte ghosts by Schatzmann [l] and its association with a calcium stimulated, magnesium dependent ATPase [(Mg + Ca)-ATPase] [l-4], few attempts have been made to determine the stoichiometry of the calcium transport system. The lack of a specific inhibitor of transport (Mg t Ca)ATPase, such as ouabain in the case of the Na,K-ATPase associated with the Na-pump [5], made the determination of the stoichiometry difficult, since ATPase activity related to calcium transport could not be readily separated from the total ATPase activity present. Ruthenium red appeared to be a promising tool for determining the stoichiometry, as Watson et al. [6] reported that this hexavalent dye specifically inhibited (Mg + Ca)-ATPase in human erythrocyte membrane fragments without affecting the Mg-ATPase or Na,K-ATPase activities. We found that ruthenium red inhibited calcium transport to a maximum of only 60% when applied to the external side of resealed ghosts [7]. Schatzmann and Tschabold [8] reported that the lanthanides praseodymium (Pr3 +) and holmium (Ho33 inhibited 100% of the active calcium transport in resealed ghosts at 1 mM concentration. Weiner and Lee [9] found that low concentrations of La3’ also specifically inhibited Ca-activated ATPase activity in membrane fragments without affecting either Mg-ATPase or Na, K-ATPase activities. In this communication we show that whereas 0.1 mM La3+ completely blocked Ca efflux when applied on the external side of resealed human erythrocyte ghosts, La3’ inhibited only 50% of the total Mgand

Calcium transport in human erythrocytes: Separation and reconstitution of high and low Ca affinity (Mg + Ca)-ATPase activities in membranes prepared at low ionic strength☆

Abstract Human erythrocyte membranes obtained by freeze-thawing of ghosts prepared in the absence or presence of EDTA, by washing with a 12 mosm medium at pH 7.7 or a 2 mosm medium at pH 6.5 contain both high and low Ca affinity (Mg + Ca)-ATPase activities. Incubation of ghosts in a less than 2 mosm medium at pH 7.5 or in 0.1 m m EDTA + 1 Him Tris-maleate (pH 8.0) results in removal of the high affinity (Mg + Ca)-ATPase activity from the membrane in a time dependent manner. Under similar conditions up to 25% of membrane proteins are removed. The soluble protein fraction extracted, although devoid of ATPase activity, reconstitutes with the remaining membrane residue with restoration of original (Mg + Ca)-ATPase activity. Addition of the soluble protein fraction to heat-treated membranes devoid of low affinity (Mg + Ca)-ATPase activity allows reconstitution of more than 33% of the original high affinity (Mg + Ca)-ATPase activity which has a Ca dissociation constant of approximately 1.6μ m . Temperature and phospholipase A 2 studies indicate that low affinity (Mg + Ca)-ATPase activity is phospholipid dependent in contrast to high affinity (Mg + Ca)-ATPase activity. Ruthenium red and LaCl 3 inhibit both high and low affinity (Mg + Ca)-ATPase activities with similar potencies. The ease of removal of high affinity (Mg + Ca)-ATPase activity from the membrane by relatively mild conditions suggests that an activator protein or the high affinity (Mg + Ca)-ATPase itself is only loosely attached to the membrane. These studies show that low affinity (Mg + Ca)-ATPase activity is not an artifact and is distinct from high affinity (Mg + Ca)-ATPase activity. The low affinity (Mg + Ca)-ATPase activity is sensitive to Ca 2+ in the concentration range from below 0.3 μ m to 300 μ m compatible with an association of this enzyme with Ca transport.

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease☆

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.

The role of tightly bound phospholipid in the activity of erythrocyte acetylcholinesterase

Summary Acetylcholinesterase was extracted from bovine erythrocyte ghosts in a hypotonic (25 m0sm) phosphate buffer (pH 7.4) and solubilized with Lubrol WX (2 mg/ml). Solubilization resulted in a decrease of the partial specific volume of the acetylcholinesterase from 0.895 ml/g in the particulate fraction to 0.793 ml/g in the Lubrol WX solubilized fraction. Both the particulate fraction and the solubilized fractions gave Arrhenius plots with a discontinuity in the activation energy at 20°C. Phospholipase A 2 or C did not abolish the discontinuity. Treatment with 1.77 M NaCl resulted in a linear Arrhenius plot of intermediate activation energy. The high salt treatment removed phospholipid not previously extracted by chloroform/methanol. The extracted phospholipid corresponded to cardiolipin on one and two dimensional silica gel chromatography. Addition of cardiolipin to a high salt-treated preparation of acetylcholinesterase restored the discontinuity in the Arrhenius plot. It is concluded that the physical state of cardiolipin, tightly bound to acetylcholinesterase by ionic interactions, modulates the catalytic activity of acetylcholinesterase.

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-.

Properties of A (Mg2+ + Ca2+)-dependent ATPase of bovine brain cortex Effects of detergents, freezing, cations and local anesthetics

Abstract 1. 1. Microsomal fractions from bovine brain cortex contain a ouabain-insensitive, Mg2+-dependent ATPase activity which is stimulated by low concentrations of CaCl2 (65 μM) [(Mg2+ + Ca2+)-ATPase]. 2. 2. NaCl or KCl (100 mM) further activates the (Mg2+ + Ca2+)-ATPase by 67%. 3. 3. Sodium deoxycholate and lubrol-WX treatment of the microsomal fraction enhances the (Mg2+ + Ca2+)-ATPase activity. This enhancement is less than that of (Na+, K+)-ATPase activity but greater than that of the basal Mg2+-ATPase activity. 4. 4. The Ca2+-stimulated component of the ATPase activity is extremely labile, Ca2+ activation reaches a maximum 2 to 4 days after storage at −12 °C, but is greatly reduced after 7 days. 5. 5. The presence of Ca2+ (65 μM) lowers the K m for ATP from 240 to 40 μM. 6. 6. Preparations containing an active (Mg2+ + Ca2+)-ATPase have a ouabain-insensitive, Mg2+-dependent p- nitrophenylphosphatase activity which is inhibited by CaCl2 and an I 50 of 65μM. 7. 7. Neither adrenergic agonists nor α-adrenergic antagonists have effects on the (Mg2+ + Ca2+)-ATPase. Tetracaine hydrochloride inhibits Ca2+ stimulation of the ATPase with an I 50 of 3.1 mM and shifts the optimum Ca2+ concentration to 178 μM. 8. 8. Since it has been suggested that the function of a (Mg2+ + Ca2+)-ATPase may be to maintain low intracellular levels of Ca2+ and perhaps excitability of membranes in nervous tissue, the inhibition of (Mg2+ + Ca2+)-ATPase by local anesthetics may account at least in part for their mechanism of action.

Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca2+-ATPase☆

The membrane-bound form and a solubilized and purified form of the Ca2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified Ca2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca2+-ATPase from proteolytic activation (K.K.W. Wang, A. Villalobo, and B.D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696-704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca2+-ATPase was proteolyzed by calpain at a slower rate.

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.

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.