The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase

Abstract

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver

“…3). This stimulation was much higher than was observed previously [18]. Substitution of EGTA for EDTA also stimulates PAP activity in the substrate that contained the higher concentrations of Ca2+, but it had no significant effect with the Ca2+-depleted substrate (Fig.…”

“…Previous work had demonstrated that chlorpromazine could stimulate PAP activity and that it might replace the requirement for Mg2+ [18]. We therefore examined whether this applied under the present assay conditions with phosphatidate that contained the higher or lower concentrations ofCa2+.…”

“…In liver [18][19][20], adipose tissue [21,22], lung [23] and other tissues, the cytosolic PAP exhibits a fairly high stimulation with Mg2+. By contrast, PAP activity in microsomal and in other particulate fractions is increased to a smaller extent by Mg2+ [21,23,24].…”

Problems encountered in measuring the activity of phosphatidate phosphohydrolase

“…The presence of tightly bound Ca2+ in the phosphatidate used to prepare the emulsion is particularly inhibitory towards the soluble enzyme . Provided that Ca2+-free phosphatidate is used and its physical state is carefully controlled, then it can be efficiently degraded by the soluble enzyme (Hosaka et al, 1975;Bowley et al, 1977;Savolainen, 1977).…”

Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity

“…Mn 2ϩ was equally effective compared with Mg 2ϩ for the binding of lipin-1 to PP-1c␥, whereas Ca 2ϩ was less effective. Lipin-1 and PP-1c both require Mg 2ϩ or Mn 2ϩ for their respective catalytic activities (23,39,40). Moreover, the interaction of lipin-1 with PP-1c␥ is mediated through a conserved HVRF motif on lipins, which closely resembles the canonical RVXF motif present on all PP-1c regulatory proteins.…”