Kinetic Characterization and Partial Purification of the Membrane-Bound Inorganic Pyrophosphatase fromRhodopseudomonas palustris

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Abstract
A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2.sym., and the (Mg-PPi) complex is regarded to be the functional substrate. Free 2.sym., revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2.sym.. Mg2.sym. is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2.sym. revealed a non-competitive type of inhibition on the Mg2.sym.-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2.sym., had to be applied. An enrichment method along with stepwise lowering the concentrations of Tritox X-100 and Mg2.sym. after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2.sym. was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity.