Effect of heat treatment on the ATPase activity of various sarcoplasmic reticulum preparations

Summary Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum (SR) preparations is activated after a short period of preincubation at temperatures between 40 and 45°C, but for temperatures higher than 48°C pronounced denaturation is observed. Heat denaturation is decreased if Mg²⁺ or K⁺ are present during heat treatment.Acknowledgments. This work was supported by research grants from Instituto de Alta Cultura (Grant No. CB/2) and the Calouste Gulbenkian Foundation. We are grateful to Drs.A. P. Carvalho andV. M. C. Madeira for many discussions and suggestions during the course of this work.     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione

Alcian blue and plumbagin induced transient Ca²⁺ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca²⁺ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca²⁺. Mg²⁺ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca²⁺ release. These results suggest that oxidation of sulfhydryls on Ca²⁺ release channels induces Ca²⁺ release even in the presence of GSH in situ.     

Symbioramide,a novel Ca2+-ATPase activator from the cultured dinoflagellateSymbiodinium sp.

Summary A novel sphingosine derivative, symbioramide, has been isolated from the laboratory-cultured dinoflagellateSymbiodinium sp. as a sarcoplasmic reticulum (SR) Ca²⁺-ATPase activator, and its structure elucidated to be1 on the basis of spectral and chemical means.Acknowledgments. We thank Ms M. Hamashima and Ms A. Muroyama for their technical assistance.     

Plakorin,a potent Ca2+-ATPase activator from the Okinawan marine spongePlakortis sp.

Summary A new cyclic peroxide, plakorin, which is a potent sarcoplasmic reticulum (SR) Ca^a+-ATPase activator has been isolated from the Okinawan marine spongePlakortis sp., its structure was elucidated on the basis of spectral data.Acknowledgments. We thank Ms M. Hamashima and Ms A. Muroyama for their technical assistance.     

Effects of various inhibitors and 2,4-dinitrophenol on adenosine triphosphatase from Malpighian tubules ofLocusta migratoria L

Summary Both Mg²⁺-ATPase and HCO ₃ ^– -stimulated ATPase activity were inhibited by sodium azide and to a lesser extent ethacrynic acid and amiloride. 1 mM DNP stimulated Mg²⁺-ATPase activity by 22% and HCO ₃ ^– -stimulated ATPase activity by 7%.     

The (Na++K+)-ATPase activity in brain of quaking mice

Summary The (Na⁺+K⁺)- and Mg²⁺-dependent ATPase distribution in several brain areas has been investigated in Quaking mutant mice characterized by myelin deficiency. A marked decrease of (Na⁺+K⁺)-ATPase activity has been found in limbic structures, hypothalamus and cerebellum. The Mg²⁺-dependent activity did not change. A possible involvement of the impairment of the (Na⁺+K⁺)-ATPase activity in the seizure susceptibility of this mice is discussed.Chargée de Recherche au CNRS.     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Insulin effect in vitro on human erythrocyte plasma membrane

Summary The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na⁺–K⁺) and Mg²⁺-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na⁺–K⁺)-ATPase activity, and at the same time decreases membrane fluidity.This investigation has been supported by Consiglio Nazionale delle Richerche, Rome, Italy.     

Solubilization and characterization of a ouabain-sensitive protein from transverse tubule membranejunctional sarcoplasmic reticulum complexes (TTM-JSR) in cat cardiac muscle

Summary A new glycoprotein of 31,500 dalton, which has a high affinity for ouabain, and is independent of (Na⁺–K⁺)-ATPase, was solubilized from transverse tubule membrane and junctional sarcoplasmic reticulum complexes (TTM-JSR) of cat cardiac muscle. This protein could be extracted only in small amounts from sarcolemmaplasma membrane (SLM-PL) fragments, suggesting that it indeed originates from the TTM-JSR.     

Temperature acclimation of Mg2+Ca2+-myofibrillar ATPase from a cold-selective teleost,Salvelinus fontinalis: a compromise solution

Summary Brook trout (Salvelinus fontinalis, Mitchill) were acclimated over 15 weeks to either +4°C or +24°C. The effects of temperature on myofibrillar Mg²⁺Ca²⁺-ATPase activities were investigated. In contrast to goldfish, temperature acclimation does not alter the kinetic properties of the brook trout myofibrillar ATPase. Activation energy (G^#) is lower and substrate turnover number is higher than values previously reported for cold-adapted stenotherms. Properties of brook trout ATPase appear to be a compromise enabling function across a broad temperature range. The different strategies of adapting to seasonal temperature variations are briefly discussed.The authors are grateful to the Wellcome Trust for financial support.-Correspondence should be addressed to I.A.J.     

Comparative analysis of phospholamban phosphorylation in crude membranes of vertebrate hearts

Summary Phospholamban, a sarcoplasmic reticulum phosphoprotein, is present in the hearts of mammalian, avian, amphibian, and fish species. Phylogenetic changes are indicated by marked differences among species in cardiac phospholamban content and by the absence of Ca²⁺/calmodulin-dependent phospholamban phosphorylation at an early developmental stage.     

9-Methyl-7-bromoeudistomin D,a potent inducer of calcium release from sarcoplasmic reticulum of skeletal muscle

Summary The Ca²⁺-releasing action of several derivatives of eudistomin D isolated from a marine tunicate was compared with that of caffeine. It was found that 9-methyl-7-bromoeudistomin D was approximately 1000 times more potent than caffeine in causing Ca²⁺ release from the sarcoplasmic reticulum.The authors thank Ms A. Muroyama of this institute for her technical assistance and Prof. K. L. Rinehart of University of Illinois for his encouragement.     

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes

Summary The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na⁺–K⁺)-ATPase and Mg²⁺-ATPase activities but not 5-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.This investigation was partially supported by the Italian National Research Council.     

Mg2+-ATPase defective mutant ofEscherichia coli and thiamine transport

Summary Mg²⁺-ATPase deficient mutant ofEscherichia coli showed an evident dependency of thiamine uptake on the oxidative metabolism of glucose, whereas the parent strain did not. In both cells, this uptake was completely inhibited by H⁺ conductors.Acknowledgment. We are indebted to Miss M. Abe for her excellent technical assistance.     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

Response of briefly glycerinated smooth muscle to Ca2+ and Mg2+

Summary Smooth muscle, treated with 50% glycerol solution at 27°C for 20 min, contracted on the application of Ca²⁺ or Mg²⁺. The briefly glycerinated smooth muscle can be used as a model system of smooth muscle contraction.     

Symbioramide, a novel Ca2+-ATPase activator from the cultured dinoflagellate Symbiodinium sp

A novel sphingosine derivative, symbioramide, has been isolated from the laboratory-cultured dinoflagellate Symbiodinium sp. as a sarcoplasmic reticulum (SR)Ca2+-ATPase activator, and its structure elucidated to be 1 on the basis of spectral and chemical means.     

Effects of Mn2+ and Mg2+ on the pigeon liver pyruvate kinase

Riassunto Mn²⁺ and Mg²⁺ attivano la piruvato cinasi di fegato di piccione in maniera distinta. In presenza di basse concentrationi di fosfoenolpiruvato Mn⁺ é piú efficace di Mg²⁺ ed é attivatore dell'enzima saturato da Mg²⁺. Piruvato cinasi (EC 2.7.1.40).

---

---

This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma, Italia.Silvia Baldi is a fellow of the Italian C.N.R.