CONTROLLED PROTEOLYSIS MIMICS THE EFFECT OF FUSICOCCIN ON THE PLASMA-MEMBRANE H-ATPASE()

We analyzed the effects of controlled treatments with trypsin of plasm a membrane (PM) isolated from radish (Raphanus sativus L.) seedlings o n the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, marke dly increased the H+-ATPase activity. The effect strongly increased wi th the increase of pH of the assay medium from 6.1 to 7.5, so the pH o ptimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the po rtion of PM H+-ATPase activity that is stable to preincubation in assa y medium in the absence of ATP and determined a strong increase of V(m ax) and a less marked decrease of the apparent K(m) for Mg-ATP. All of these effects were very similar to those determined by FC, which acti vated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attac k of trypsin. Conversely, trypsin treatment had little effect on the P M H+-ATPase preactivated with FC. Moreover, the activity of the PM H+- ATPase preactivated with FC was not further activated by lysolecithin. These results indicate that the modification of the PM H+-ATPase of h igher plants triggered by the FC-receptor complex hinders the inhibito ry interaction of the regulatory C-terminal domain with the active sit e.