Subunit composition and Ca(2+)-ATPase activity of the vacuolar ATPase from barley roots.
The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.
DuPont
F M
FM
U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California 94710.
Morrissey
P J
PJ
D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D010944:Plants / Q000201:enzymology; D011092:Polyethylene Glycols; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D003902:Detergents; D046911:Macromolecular Substances; D011092:Polyethylene Glycols; D017830:Octoxynol; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium