Purified vacuolar inorganic pyrophosphatase consisting of a 75-kDa polypeptide can pump H+ into reconstituted proteoliposomes

• Published in: The Journal of biological chemistry Vol. 269; no. 9; pp. 6725 - 6728
• Main Authors: Sato, M.H, Kasahara, M, Ishii, N, Homareda, H, Matsui, H, Yoshida, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 04.03.1994
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology; CATION; CATIONES; Chromatography, Ion Exchange; CUCURBITA; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; FISIOLOGIA VEGETAL; Fundamental and applied biological sciences. Psychology; HIDROGENO; HIDROLASAS; Hydrogen-Ion Concentration; HYDROGENE; HYDROLASE; Hydrolases; Inorganic Pyrophosphatase; Intracellular Membranes - enzymology; ION; IONES; Kinetics; LIPOSOMAS (ORGANULOS); LIPOSOME (ORGANITE); Liposomes; Magnesium - pharmacology; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; Membrane Potentials - drug effects; Membrane Potentials - physiology; Microscopy, Electron; Molecular Sequence Data; Molecular Weight; PHYSIOLOGIE VEGETALE; Potassium - metabolism; Potassium Chloride - pharmacology; Proteolipids - metabolism; Pyrophosphatases - isolation & purification; Pyrophosphatases - metabolism; Pyrophosphatases - ultrastructure; Sequence Homology, Amino Acid; VACUOLA; VACUOLE; Vacuoles - enzymology; Vegetables - enzymology; Enzyme; Cucurbitaceae; Ion transport; Electron microscopy; Vacuole; Inorganic pyrophosphatase; Proteoliposome; Cucurbita; Membrane transport; Dicotyledones; Angiospermae; Proton; Hydrolases; Spermatophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37435-5

Inorganic pyrophosphatase was purified to homogeneity from the vacuolar membranes of pumpkin hypocotyl tissue. The purified Inorganic pyrophosphatase consists of a single kind of 75-kDa polypeptide, and the stacked molecules with a repeating unit of 5.8 X 3.8 nm are seen under electron micrography. It exhibits H+-translocating activity across membranes coupled with PPi hydrolysis when it is reconstituted into proteoliposomes. A monovalent cation is required for the H+ translocation with the K+ ion being the most effective, but evidence for active transport of 42K+ into proteoliposomes was not obtained under the conditions tested. The hydrolysis of PPi by the reconstituted proteoliposomes is stimulated by the addition of a H+ ionophore, carbonyl cyanide p-trifluoromethyoxyphenylhydrazone, but not by a K+ ionophore, valinomycin. Both hydrolysis of PPi and PPi-dependent H+ translocation of the proteoliposomes are inhibited by N,N'-dicyclohexylcarbodiimide