Enzymatic properties of phosphatidylinositol‐glycan‐specific phospholipase C from rat liver and phosphatidylinositol‐glycan‐specific phospholipase D from rat serum

FEBS Journal - Tập 197 Số 1 - Trang 67-73 - 1991
S. Stieger1, Susanna Diem2, A. Jakob2, Urs Brodbeck1
1Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
2Biochemisches Institut, Universität Basel, Switzerland

Tóm tắt

Using anchored acetylcholinesterase from bovine erythrocytes and phosphatidylinositol‐glycan (PtdIns‐glycan) as substrate, we found PtdIns‐glycan‐anchor‐degrading activity in rat liver and serum. 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 enzymes 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 μM and 0.16 μM, 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 μM 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.

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FEBS Journal

Tập 197 Số 1

67-73

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