Purified vacuolar inorganic pyrophosphatase consisting of a 75-kDa polypeptide can pump H+ into reconstituted proteoliposomes.
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.
Sato
M H
MH
Research Laboratory for Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Kasahara
M
M
Ishii
N
N
Homareda
H
H
Matsui
H
H
Yoshida
M
M
D000595:Amino Acid Sequence; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism*; D011755:Pyrophosphatases / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology*; D014675:Vegetables / Q000201:enzymology*
D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D011189:Potassium Chloride; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D011188:Potassium