Purification and characterization of a divalent cation-activated ATP-ADPase from pea stem microsomes.

The microsomal fraction (13 000 -60 000 x g pellet) from pea stem shows a very high divalent cation-dependent, diethylstilbestrol- and orthovanadate-inhibited ATPase and ADPase activity. No detectable inorganic or organic pyrophosphatase and adenylate kinase and almost negligible activities of mitochondrial ATPase and of other phosphomonoesterases are present in this preparation. The ATPase and ADPase activities of microsomes have been solubilized with NaClO4 and then purified by gel filtration and DEAE-Sephadex fractionation to a final specific activity of 71.5 and 102 mumol Pi/min per mg for ATP and ADP, respectively. The purified enzyme hydrolyzes triphosphonucleosides (ATP, CTP, GTP, UTP) and diphosphonucleosides (ADP and to a lesser extent CDP, UDP, IDP) and presents pH optima of 6 for ATP and 7 for ADP. It requires Mg2+, Mn2+ or Ca2+ and is inhibited by diethylstilbestrol and orthovanadate. The conclusion that the ATPase and ADPase activities belong to the same enzyme is based on the following results: (1) parallel effects of diethylstilbestrol and orthovanadate on both ATPase and ADPase; (2) parallel behavior of ATPase and ADPase throughout all the purification steps; (3) non-additivity of ATPase and ADPase and (4) lack of dilution of beta-32P formed from [beta-32P]-ATP by unlabelled ADP.