Effect of the photoperiod on the plasma membrane ATP-dependent H + -pumping activity of spinach petioles

Abstract

Effect of the photoperiod on the plasma membrane ATP-dependent H+-pumping activity of spinach petioles. The plasma membrane from spinach (Spmac/a o/eracea, cv.Nobel) petioles was purified by phase partitioning and the use of enzyme markers showed that this fraction was highly enriched in plasma membrane vesicles. This fraction was almost devoid of phosphohydrolase activities originating from endomembranes (tonoplastes, mitochondria, non specific phosphatases and golgi apparatus). The ATP-dependent H+ pumping activity in this fraction was Mg^+ and K+ dependent and was not stimulated by valinomycin. It was unsensitive to nitrate (tonoplaste ATPase inhibitor) but sensitive to vanadate (plasma membrane ATPase inhibitor). This activity was specific for ATP and had a pH optimum around 6.5. The kinetic constants (Km.,pp and for ATP) of the H+ pumping activity of the purified plasma membrane were determined from plants grown in short days (vegetatives) and from similar plants induced by 24 h of continuous light. Whereas Km have not been changed after light induction, values of and the activity in the presence of 0.5 mB ATP have been increased suggesting a regulation of the enzyme after light induction. Key-words: H+-ATPase, Plasma membrane, Sp/rtac/a o/eracea. Abbreviations: ADP. Adenosine diphosphate; ATP, Adenosine triphosphate; BSA, bovine serum albumin; BTP, bis-tris-propane (1.3-bis (tris (hydroxymtehyl) methylamino) propane); DTT, dithiothreitol; GTP. Guanosine triphosphate; 1DP, inosine diphosphate; Mes. 4-morpholinoethane sulfonic acid; PNPP, p-nitrophenylphosphate; PMSF, phenylmethylsulfonylfluoride: PPj, Pyrophosphate; Tris, Tris (hydroxy methyll-aminomethane; UDPG, uridine diphosphoglucose.