Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate magnesium

Lineweaver-Burk plots of Ca²⁺-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg²⁺ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg²⁺ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg²⁺.     

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast

1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.     

A study of the interactions among adenosine triphosphate, epinephrine, and magnesium ions

The interactions among adenosine triphosphate, Mg⁺², and epinephrine at pH's below 7.0 have been studied by observing the effects of these interactions on the chemical shifts and line widths of their ¹H and ³¹P nuclear magnetic resonance spectra. Mg⁺² is tightly bound by the β- and γ-phosphate groups of adenosine triphosphate and there is a weak association between this chelate and epinephrine. In the ternary complex, the aromatic ring of epinephrine overlaps the purine ring of adenosine triphosphate and there appears to be an ionic interaction between the protonated amino group and the α-phosphate of adenosine triphosphate. It was also found that dichloroisoproterenol forms essentially the same type of ternary complex.     

Effects of caffeine, calcium and magnesium on plant cytokinesis

The study of the cytokinesis inhibition by caffeine in meristem cells of onion root tips has shown the antagonism between calcium and/or magnesium and caffeine. Moreover, the influence of chelating agents (citrate and EDTA), which potentiate the caffeine effect on cytokinesis, also suggest an essential role for both cations in this process. We propose that caffeine interferes with plant cytokinesis involving some aspect of membrane recognition and/or fusion, where calcium and magnesium are essential requirements.     

Effect of magnesium and calcium ions on blocking of synaptic transmission by adenosine in isolated rat hippocampal slices

Dependence of the inhibitory action of adenosine on the extracellular composite EPSP on the concentrations of Mg and Ca cations in the medium was investigated in isolated slices of rat hippocampusin vitro. Extracellular EPSPs were derived in the region of apical dendrites of pyramidal cells in area CA₁ during stimulation of Schaffer's collaterals. The blocking action of bivalent cations (an increase in Mg⁺⁺ or a decrease in Ca⁺⁺) developed almost five times more slowly than the action of adenosine. An increase in the external Mg⁺⁺ concentration potentiated whereas a decrease weakened the inhibitory action of adenosine. Ca⁺⁺ ions had the opposite effect. Antagonistic relations were exhibited between Mg⁺⁺ and Ca⁺⁺ ions. Analysis of dose-response curves for adenosine showed that during a simultaneous increase in the extracellular Ca⁺⁺ and decrease in Mg⁺⁺ concentrations, not only was the maximal effect of adenosine reduced, but so also was its binding constant with the receptor. The results suggest that antagonism between Ca cations and adenosine is mixed in character — both competitive and noncompetitive. The possible mechanism of the inhibitory action of adenosine on synaptic transmission and the role of bivalent cations in this process are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 532–539, July–August, 1984.     

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.     

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.     

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.     

Inhibition of beta-amylase activity by calcium, magnesium and zinc ions determined by spectrophotometry and isothermal titration calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH = 4.8 (sodium acetate 16 mM) and T = 300K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.     

Inhibition of smooth muscle 5'-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5'-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions.