Properties of the partially purified tonoplast H+-pumping ATPase from oat roots.
Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.
Randall
S K
SK
Sze
H
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002850:Chromatography, Gel; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D002523:Edible Grain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D017830:Octoxynol; D010743:Phospholipids; D011092:Polyethylene Glycols; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D012995:Solubility; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D001386:Azides; D009566:Nitrates; D010743:Phospholipids; D014639:Vanadium; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D014638:Vanadates; D011092:Polyethylene Glycols; D004024:Dicyclohexylcarbodiimide; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017830:Octoxynol; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid