Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase.
The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.
Manolson
M F
MF
Rea
P A
PA
Poole
R J
RJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D002234:Carbodiimides; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases