The uptake and internal distribution of solutes, driven by ion-translocating ATPases, is one of the major ATP consuming processes in the eucaryotic cell. In Neurospora crassa, as in many other kinds of eucaryotic cells, the plasma membrane ATPase functions as a proton pump. In its structure and reaction mechanism, however, this H ion-ATPase is representative of cation-translocating enzymes such as the (Na ion, K ion)-ATPase and the Ca2 ion-ATPase of animal cells. Having succeeded in solubilizing and purifying the plasma membrane ATPase of Neurospora, we now propose to undertake a detailed analysis of its reaction mechanism. In particular, we will rigorously characterize the kinetic behavior of the enzyme by measuring effects of Mg, ATP, and vanadate on its activity and by in vitro binding studies with vanadate (as 48V). In addition, with newly developed genetic approaches, we plan to compare genetically altered ATPases with the wild type enzyme to probe the structure and function of the ATPase. An intracellular ATPase, shown to be present on vacuolar membranes of fungi, plants, and animal secretory cells, is postulated to be a proton pump and is expected to share the cytoplasmic pool of ATP with the plasma membrane ATPase. Using a highly purified preparation of Neurospora vacuoles, we have preliminary evidence for a unique type of ATPase in these organelles. We propose to characterize the vacuolar membrane ATPase, to purify it, and to determine if its properties are consistent with its postulated role as a proton pump. By comparing the reaction mechanism and kinetic regulation of the plasma membrane and vacuolar enzymes, we hope to explore the ways in which coordinate regulation of two ATPases, sharing a cytoplasmic pool of ATP, is achieved.