A model for regulation of the Mg(2+)-stimulated acto-myosin-ATPase activity: inhibition of the formation of actin-myosin complex and the Mg( 2+)-stimulated acto-myosin-ATPase activity by IMP and AMP

Previously, we showed that the decrease in force output during continuous isometric contractions in rat skeletal muscle was related to an increase in the concentration of IMP. In this paper we report on additional experiments in which the effect of IMP on the Mg(2+)-stimulated acto-myosin-ATPase activity of isolated actin and myosin is measured at 35 degrees C. The results show that 1) the binding of actin to myosin is co-operative (Hill coefficient = 3.82); 2) in the presence of IMP or AMP the Mg(2+)-stimulated acto-myosin-ATPase activity is inhibited up to 60% at 10 mM; 3) in the presence of IMP or AMP not only the Mg(2+)-stimulated acto-myosin-ATPase activity decreases, but also K(50). From these results we conclude that IMP and AMP may be considered as uncompetitive inhibitors. Our results suggest that IMP and AMP can prevent an 'energy crisis' during exhaustive exercise of short duration by down-regulating the contractile machinery.     

Mg2+ inhibition of Na2+-stimulated Ca2+ release from brain mitochondria

Magnesium has been shown to modulate the Na+-stimulated release of Ca2+ (Na/Ca exchange) from brain mitochondria. The presence of 5 mM MgCl2 extramitochondrially inhibits the Na/Ca exchange as much as 70%. Additionally, Na+-stimulated Ca2+ release is enhanced by the presence of divalent chelators, this stimulation also being inhibited by the addition of excess Mg2+. The inhibitory effect of Mg2+ and the enhancement by chelating agents were both reversible. Heart mitochondria exhibit a similar enhancement of Na/Ca exchange by chelators and inhibition by MgCl2, though not as pronounced.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.     

The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes.

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.     

Adenylate cyclase inhibition by hormones. The Mg2+ hypothesis

In washed anterior pituitary membranes, there is enough GTP to occupy Ns and therefore to obtain activation of adenylate cyclase by vasointestinal peptide. GTP concentrations needed to obtain adenylate cyclase inhibition by dopamine (above 5 X 10- M) stimulate the adenylate cyclase. The dopamine effect is a blockade of this stimulation. We propose that at least in this system, Ni does not inhibit but stimulates the adenylate cyclase and that inhibitory hormones block this stimulation. We also demonstrate in several adenylate cyclase systems that hormones produced adenylate cyclase inhibition by lowering their Mg affinity A general model for adenylate cyclase activation and inhibition is proposed.     

Na+-like effect of monovalent cations in the stimulation of sea bass gill Mg2+-dependent Na+-stimulated ATPase

The Mg2+-dependent ouabain insensitive-ATPase activity present in gill microsomal preparations from Dicentrarchus labrax is stimulated not only by Na+ but also by K=, NH4+ or Li+. These cations at 50-100 mM concentrations are similarly efficient to Na+ in stimulating the enzyme activity with similar Km values. Whatever cation stimulates the activity, the enzyme is poorly sensitive to ouabain and 100% inhibited by 1.5-2.5 mM ethacrynic acid. All activity vs cation concentration curves show a biphasic profile with activation following the Michaelis-Menten kinetics (Hill coefficient approximately 2). The absence of additivity when the enzyme is activated by binary mixtures of cations, each of which may act as competitive inhibitor of the other confirms the involvement of the same binding site for the monovalent cations.     

High-affinity Ca2+-stimulated and Mg2+-dependent ATPase from rat osteosarcoma plasma membranes

Transplantable rat osteosarcoma plasma membrane preparations contain high-affinity and low-affinity calcium-stimulated ATPases. The high-affinity enzyme displayed a K0.5 for calcium of 0.03 microM, a Vmax of 99.2 nmol/min/mg, and a requirement for magnesium ions. It was not inhibited by 20 microM trifluoperazine nor stimulated by the addition of 2 ng of calmodulin. Lack of stimulation with exogenous calmodulin may be related to the high endogenous calmodulin content of the membrane preparations. The low-affinity Ca2+- or Mg2+-ATPase displayed a K0.5 for calcium of approximately 2.40 mM (Vmax of 185 nmol/min/mg) and a K0.5 for magnesium of approximately 2.75 mM (Vmax of 250 nmol/min/mg).     

The effect of Mg2+ and Ca2+ on urea-catalyzed phosphorylation reactions

Summary When struvite (MgNH₄PO₄ 6H₂O) is heated with urea at 65–100°C, inorganic pyrophosphate is formed in good yield. Under similar conditions pyro-phosphate is formed much more slowly from ammonium phosphate or hydroxylapatite. The major products formed by the reaction of nucleotides with urea and either ammonium phosphate or hydroxylapatite are derivatives phosphorylated on the 2 or 3 position. With struvite, on the other hand, the main reaction is pyrophosphate bond formation. Yields of up to 25% of uridine diphosphate can be obtained at temperatures as low as 65°C.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts.

Myosin purified from a murine myeloid leukaemia cell line (M1) that had been incubated with [32P]orthophosphate incorporated 32P into the heavy, but not the light, chain. When the heavy chain was dephosphorylated by bacterial alkaline phosphatase, myosin that had low actin-activated ATPase activity gained higher activity only in the presence of the light-chain kinase. In the absence of the light-chain kinase, however, the Mg2+-stimulated ATPase activity of myosin was not activated by actin, regardless of phosphatase treatment. These results indicate that the activity of M1 myosin ATPase is regulated by phosphorylation of both the light and heavy chains. A scheme for this regulation by phosphorylation is presented and discussed.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH

Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.     

Actin-stimulated myosin Mg2+-ATPase inhibition by brain protein

A low-molecular-weight protein, isolated from bovine brain, inhibits the actin-stimulated Mg-ATPase activity of myosin from striated muscle. This inhibition is probably related to its ability to cause actin to revert from its polymerized to its depolymerized state and to prevent the polymerization of actin. Its effect on the polymeric state of the actin has been demonstrated by viscosity studies. DNase inhibition assay, and electron microscopy. Heavy meromyosin can overcome the effect of the brain protein and stimulate the polymerization of actin. The inhibition of ATPase activity is in part dependent upon the relative amounts of brain protein, actin, and myosin. The apparent molecular weight of the brain protein is approximately 20,000 daltons. It appears to be a heat-labile glycoprotein containing 5-6% carbohydrate.     

Cytoplasmic pH and free Mg2+ in lymphocytes

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly- trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced no net movement of Mg2+ or H+; (b) by Mg- sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.     

The effect of temperature,Ca2+, Mg2+ and Ni2+ ions on the swimming speed of C. reinhardii determined by quasi-elastic light scattering

Quasi-elastic light scattering was used to measure the motility of Chlamydomonas reinhardii (wild type) under varying environments of temperature and ionic concentration (Ca²⁺, Mg²⁺, Ni²⁺). The results obtained agree with the results obtained by other techniques where comparison was available. The advantage of this method is that it provides these results in minutes. In many cases this rapid analysis can be used to follow the change in motility which arises after a chemical or physical perturbation. The speed of the cell, and the average flagellar force, was found to increase as the temperature was varied between 6 and 40 °C. The cell seemed to be irreversibly damaged at temperatures above 40 °C. The speed of the cell was found to be very dependent on [Ca²⁺]. The cell's speed slowed very significantly as the concentration of Ca²⁺ was reduced and was also found to be relatively independent of the Mg²⁺ concentration between 10^?3 and 10^?10 M. The addition of Ni²⁺ to the cells' environment resulted in the cell speed increasing significantly over the short term, with the long-term effect being a net decrease in the cells' speed.     

Regulation of the microtubule-lysosome interaction: activation by Mg2+ and inhibition by ATP

We developed a sedimentation assay to characterize and quantify the association of purified lysosomes to reconstituted microtubules (Mithieux, G., Audebet, C. and Rousset. B. (1988) Biochim. Biophys. Acta 969, 121-130). In the present work, we have examined the potential regulatory role of ATP and Mg2+ on the microtubule-lysosome interaction. The formation of microtubule-lysosome complexes takes place in the absence of Mg2+, but is activated by the addition of Mg2+; both the rate of the interaction and the amount of complexes formed are increased. The maximal effect is observed between 1.5 and 3.5 mM free Mg2+. Measured at the plateau of the interaction, the proportion of microtubules bound to lysosomes increases as a function of free Mg2+ concentration; at optimal concentration of free Mg2+, 90% of the microtubules present in the incubation mixture are bound to lysosomes. ATP induces a concentration-dependent inhibition of the formation of microtubule-lysosome complexes. The half-maximal effect is obtained at an ATP concentration of 0.83 +/- 0.11 mM (n = 7). The effect of ATP is not related to ATP hydrolysis, since ATP exerts its inhibitory action in the presence of EDTA. The ATP effect is mimicked by GTP, p[NH]ppA and tripolyphosphate, ADP and pyrophosphate, but not by AMP or phosphate. In the presence of 1 mM ATP, a Mg2+ concentration of 3 mM (corresponding to 2 mM free Mg2+) is required to overcome the inhibition caused by ATP; above 3 mM, Mg2+ exerts its activating effect. Since the modulating effects of ATP and Mg2+ are obtained at concentrations closed to those occurring in intact cells, we conclude that the regulation of the microtubule-lysosome interaction reported in this paper could be of physiological significance.