Effect of salts on membrane binding and activity of adipocyte phosphatidate phosphohydrolase

Abstract

Isosmotic replacement of sucrose in a low ionic strength homogenizing buffer (0.25 M sucrose/1 mM EDTA/1 mM Tris-HCl, pH 7.4) with KCl increased the microsomal and decreased the soluble phosphatidate phosphohydrolase (EC 3.1.3.4) activity of isolated rat fat cells. At 54 mM KCI the microsomal specific activity was increased 6-fold and the soluble activity was decreased to less than one-third. Binding of enzyme was promoted by KCI and NaCI when the once isolated soluble and microsomal fractions were recombined and incubated at 37° C. Half-maximal binding occurred at about 17 mM salt and maximal binding at about 50 mM. The pH optimum of binding was 7.8 in 15 mM Hepes. MgCl 2, CaCl 2and spermine prevented desorption of microsomal enzyme at μ molar levels and maximal effects were observed at concentrations below the 1 mM level. At maximum, however, the prevention of desorption was less by these salts than it was by KCI. MgCl 2 and spermine also interfered with the effect of KCI. Moderate salt-induced loading of microsomes with the phosphohydrolase (specific activity increased 3.5-fold) increased their ability to incorporate 14C into triacylglycerol from sn-[U- 14C]glycerol 3-phosphate while a high loading (specific activity increased 6-fold) had no effect or even suppressed it. The results are discussed in relation to a role of translocation of phosphatidate phosphohydrolase in glyceride biosynthesis and its control.