Effect of salts on membrane binding and activity of adipocyte phosphatidate phosphohydrolase.
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 KCl 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 KCl and NaCl when the once isolated soluble and microsomal fractions were recombined and incubated at 37 degrees 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. MgCl2, CaCl2 and spermine prevented desorption of microsomal enzyme at mu 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 KCl. MgCl2 and spermine also interfered with the effect of KCl. 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.
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009994:Osmolar Concentration; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology*; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D012965:Sodium Chloride; D011189:Potassium Chloride; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase