Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts.
The dicyclohexylcarbodiimide-sensitive ATPase from spinach chloroplast has been isolated. On sodium dodecyl sulfate gels, seven different polypeptides were seen. Five of these polypeptides coincided with the CF1 subunits, a 7,500-dalton peptide was identified as the proteolipid which interacts with [14C]dicyclohexylcarbodiimide, and there was a 15,500-dalton hydrophobic polypeptide with unknown function. In two-dimentional gels, two additional peptides were resolved, one 17,500 daltons (co-migrating in sodium dodecyl sulfate gels with subunit delta) and one 13,500 daltons (co-migrating with subunit epsilon). Reconstitution was obtained by freezing and thawing the complex with a crude mixture of phospholipids. After reconstitution the complex catalyzed 32P1-ATP exchange (rates of 200 to 400 nmoles x mg-1 x min-1) and ATP formation during acid-to-base transition. These reactions were inhibited by dicyclohexylcarbodiimide and uncouplers. Uncouplers at low concentrations stimulated and at high concentrations inhibited the Mg2+-ATPase activity. ATP hydrolysis and 32P1-ATP exchange were catalyzed by the complex in the presence of either Mg2+ or Mn2+ but not with Ca2+ or Co2+. ATP and GTP were substrates for the exchange reaction but not ADP or CTP.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D002234:Carbodiimides / Q000494:pharmacology*; D002736:Chloroplasts / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010944:Plants
D002234:Carbodiimides; D008345:Manganese; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D008274:Magnesium