title: The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase.
abstract: We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002794:Choline / Q000494:pharmacology*; D005260:Female; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008871:Microvilli / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000494:pharmacology; D010740:Phospholipases / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology; D051381:Rats; D013816:Thermodynamics
chemical_list: D007527:Isoenzymes; D008081:Liposomes; D010740:Phospholipases; D000469:Alkaline Phosphatase; D010447:Peptide Hydrolases; D002794:Choline