Enzymatic properties of phosphatidylinositol-glycan-specific phospholipase C from rat liver and phosphatidylinositol-glycan-specific phospholipase D from rat serum.
Using phosphatidylinositol-glycan (PtdIns-glycan) anchored acetylcholinesterase from bovine erythrocytes as substrate, we found PtdIns-glycan-anchor-degrading activity in rat liver and serum [corrected]. The hepatic enzyme was only soluble in detergents, whereas the serum enzyme occurs as soluble, slightly amphiphilic protein. Using 3-trifluoromethyl-3-(m- [125I]iodophenyl)diazirine-labelled acetylcholinesterase as substrate, we showed that the hepatic anchor-degrading enzyme had a cleavage specificity of a phospholipase C, whereas the serum enzyme was a phospholipase D. Both enzyme exhibited maximal activity in slightly acidic conditions and at low ionic strength. They had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 0.1 microM and 0.16 microM, respectively). Both hepatic PtdIns-glycan-specific phospholipase C and serum PtdIns-glycan-specific phospholipase D gave a large increase in activity between 0.1-10 microM Ca2+, indicating that PtdIns-glycan-specific phospholipases are only marginally active at physiological intracellular Ca2+ concentrations. The enzymes were inhibited by heavy metal chelating agents such as 1,10-phenanthroline and 2,2'-bipyridyl but not by the corresponding Fe2+ complexes or non-chelating analogues, indicating that they both require a heavy metal ion for the expression of catalytic activity in addition to Ca2+. Another interesting property of PtdIns-glycan-specific phospholipases is their inactivation by bicarbonate and cyanate. The inactivation was time- and pH-dependent and could be reversed by dialysis. These observations are in agreement with a covalent modification of the enzymes by carbamoylation.
Stieger
S
S
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Diem
S
S
Jakob
A
A
Brodbeck
U
U
D000110:Acetylcholinesterase / Q000097:blood; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000097:blood* / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D002614:Chelating Agents; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase