Time-dependent inhibition of phospholipase C beta-catalysed phosphoinositide hydrolysis: a comparison of different assays.
The properties of three different beta-isoforms of phospholipase C (PLC) were analysed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles and phospholipid monolayers spread at an air-water interface. Phosphatidylinositol 4,5-bisphosphate hydrolysis went virtually to completion in monolayers, but inositol trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by increasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when < or = 10% of PtdIns(4,5)P(2) had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by addition of fresh substrate or fresh enzyme after the slow phase of activity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest that the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipic to PLC in lamellar assays, leading to reduced activity.
James
S R
SR
Department of Biochemistry, Medical Sciences Institute, University of Dundee, U.K.
Smith
S
S
Paterson
A
A
Harden
T K
TK
Downes
C P
CP
D000818:Animals; D002384:Catalysis; D003902:Detergents; D004912:Erythrocytes / Q000201:enzymology; D006868:Hydrolysis; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008322:Mammals; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014422:Turkeys; D010738:Type C Phospholipases / Q000032:analysis / Q000378:metabolism*
D003902:Detergents; D007527:Isoenzymes; D008081:Liposomes; D008823:Micelles; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D011994:Recombinant Proteins; D010738:Type C Phospholipases