Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean.
Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.
Maeshima
M
M
Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan.
Yoshida
S
S
D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D002852:Chromatography, Ion Exchange; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology; D007074:Immunoglobulin G; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010944:Plants / Q000378:metabolism*; D010946:Plants, Medicinal; D011188:Potassium / Q000494:pharmacology; D011755:Pyrophosphatases / Q000302:isolation & purification* / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology*
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D012265:Ribonucleotides; D004024:Dicyclohexylcarbodiimide; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D011188:Potassium