Membrane-bound Pyrophosphatase Research Articles

A membrane-bound pyrophosphatase from respiratory membranes of Rhodospirillum rubrum

A pyrophosphatase activity was found in respiratory membranes of Rhodospirillum rubrum. This activity was specific for pyrophosphate and was inhibited by dicyclohexylcarbodiimide, NaF and pyrophosphate analogues, but not by oligomycin or LiCl. The divalent cation selectivity was Zn2+ > Mg2+ > Co2+ > Ca2+, and the pH dependence was the same as that of the membrane-bound pyrophosphatase of chromatophores (optimum pH 6·5). The pyrophosphate hydrolysis activity of respiratory membranes was inhibited by 1-butanol. The enzyme was solubilized by Triton X-100, and the activity of the solubilized enzyme was stimulated by phospholipids. The respiratory-membrane enzyme ran in native electrophoresis with the same R F as the membrane-bound pyrophosphatase of chromatophores. Antibodies raised against both enzymes cross-reacted with each other. These findings show that a membrane-bound pyrophosphatase is present in respiratory membranes of R. rubrum and is similar to the enzyme of photosynthetic membranes.

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 Mg2+ or Pi for the enzyme; Mn2+ and Co2+ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg2+; other divalent cations like Zn2+ or Ca2+ do not support the exchange reaction. In the hydrolytic reaction, Zn2+, at low concentration, substitutes for Mg2+ as substrate, and Co2+ also substitutes in limited amount (50%). Other cations (Ca2+, Cu2+, Fe2+, etc.) do not act as substrates in complex with PPi. The Zn2+ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn2+. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn2+ greater than Mn2+ greater than Ca2+ greater than or equal to Co2+ greater than Fe2+ greater than Cu2+ greater than Mg2+. The data in this work suggest that H+ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient.

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures.

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase of Rhodospirillum rubrum: Effects of Mg 2+, phosphate, and pyrophosphate

The relation that exists between the P i- PP i exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores of Rhodospirillum rubrum was studied. The two reactions have a markedly different requirement for added Mg 2+. Optimal rates of hydrolysis were attained at 1 m m Mg 2+ with 0.67 m m pyrophosphate; the rate of hydrolysis correlated with the concentration of Mg-pyrophosphate, which indicated that the latter was the substrate for hydrolysis. The P i- PP i exchange reaction rate was low at concentrations of added Mg 2+ below 1 m m (0.67 m m pyrophosphate), but increased as the concentration of Mg 2+ in the medium was increased. The P i- PP i exchange reaction depends on the concentration of MgHPO 4, which suggests that this is the substrate in the exchange reaction. However, it is likely that free Mg 2+ also exerts a favorable effect on the P i- PP i exchange reaction. The optimal concentration for the P i- PP i exchange reaction was approx 240 μ m, which suggests that the concentration of the hydrolyzable substrates modulates the kinetic characteristics of the enzyme.

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth.

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.

A Membrane-bound Pyrophosphatase in Escherichia coli Catalyzing the Hydrolysis of Cytidine Diphosphate-Diglyceride

Abstract An enzyme discovered in the cell membrane fraction of Escherichia coli catalyzes the following reaction: CDP-diglyceride + H2O → CMP + phosphatidic acid The enzyme has been extracted from the membrane fraction with the nonionic detergent Triton X-100 and has been purified 115-fold. This novel hydrolase appears to have a high specificity, since it does not attack at a significant rate DPN, ATP, or CDP-choline, substrates that are rapidly split by nucleotide pyrophosphatase(s) of broad specificity previously described in plant and animal tissues. It also appears to be distinct from the periplasmic bacterial hydrolases specific for nucleoside diphosphate sugars. The CDP-diglyceride hydrolase is devoid of phospholipase D activity and is also unusual in that it is strongly inhibited by AMP.