Irreversible inhibition of adenosine triphosphatases, diglyceride kinase and phosvitin kinase of brain by diisopropylphosphorofluoridate.

Abstract

High activities of transport ATPase and diglyceride kinase, relative to other preparations, were observed in a deoxycholate-treated particulate fraction from guinea-pig cerebrum. Incubation of the enzyme preparation in various buffers showed an initial inactivation of the diglyceride kinase and a slight activation of the transport ATPase. Both enzymes were irreversibly inhibited on treatment with DFP. In the presence of strophanthidin both enzymes showed similar but not identical rates of inhibition by DFP. The effects of DFP concentration and pH on the inhibitions of both enzymes were similar. ATP protected both enzymes against DFP inactivation, but very much lower concentrations were required for protection of the transport ATPase. Mg 2+ was required for DFP inhibition of transport ATPase but not of diglyceride kinase, even though the latter could be shown to require Mg 2+ for activity. Strophanthidin or K + potentiated the inhibitory effects of DFP on transport ATPase but had no effect on the inhibition of diglyceride kinase. The brain enzyme preparation also contained phosvitin kinase. It was slightly inhibited by DFP during the first few minutes of incubation; no further inhibition was observed on longer incubation. Removal of a sulfhydryl inhibitor from the DFP preparation by fractional distillation did not reduce the inhibitory effect of DFP on the three enzymes. Under conditions in which the rate of inhibition of the transport ATPase by DFP was increased 2.5-fold by strophanthidin there was no increased phosphorylation of the enzyme preparation by [ 32 P]DFP. The possibility that the transport ATPase is a modified form diglyceride kinase, in which water has become the favored phosphate acceptor, is discussed. Tetraethylpyrophosphate showed about the same potency as DFP in inhibiting the transport ATPase.