Calcium-dependent phosphorylation regulates the plasma-membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H+-ATPase (EC 3.6. 1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially puri fied H+-ATPase were used to investigate the effects of Ca2+ and calmodulin on the H+-ATPase activity and on its phosphorylation status. Both the hydro lytic and the proton-pumping activities were reduced approximately 50% by m icromolar Ca2+ concentrations while calmodulin did not show any effect eith er alone or in the presence of Ca2+. The lack of effect of calmodulin antag onists indicated that calmodulin was not involved in this response. The add ition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca2+. Phosphorylation of plasma membrane and partially purified H+-ATPa se showed the same behavior. In the presence of Ca2+ polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H+-ATPase of maize roots. The autoradiogram of the immunodetect ed protein clearly showed that this polypeptide, which corresponds to the H +-ATPase, was phosphorylated. Additional clear evidence comes from the immu noprecipitation experiments: the data obtained show that the H+-ATPase acti vity is indeed influenced by its state of phosphorylation.