Hydrolysis of glyceryl tri[1-14C]octanoate and glyceryl tri[1-14C]oleate monolayers by postheparin lipolytic activity

Abstract

The hydrolysis of monomolecular films of glyceryl tri (I-W)octanoate and glyceryl tri(l-W)oleate has been demonstrated by measurement of the decrease in surface radio- activity that occurs in the presence of postheparin plasma. The hydrolysis displayed first order kinetics and was propor- tional to enzyme concentration over a 10-fold range. No hydrolysis was observed in the absence of enzyme, and only slight activity (1%) was found in plasma taken from subjects before heparin administration. The hydrolysis was stimulated to a variable extent by Ca*+. The first product of hydrolysis of the monolayer was identified as 1,2-diglyceride, which was subsequently converted to 2-monoglyceride. Inhibition of tri- glyceride hydrolysis was observed when postheparin plasma was preincubated in 2 M NaCI, 10-4 M protamine, 10 m~ Na4P207, and 0.1 M NaF. Monolayer techniques avoid some but not all of the problems associated with emulsified lipid substrates and appear to be applicable for study of post- heparin lipolytic activities. systems the enzyme catalysis takes place only at the air- water interface with lipid films of defined composition and physical state. The requirements for enzymic reac- tions involving lipid substrates may be readily deter- mined. The effects of activators and inhibitors on the enzyme, the substrate, and the enzyme-substrate com- plex may thus be differentiated and properly evaluated. The initial objective of the series of experiments presented in this report has been to demonstrate that hydrolysis of triglyceride monolayers can be observed with the postheparin lipolytic activity (8). The mono- layer techniques appear to be applicable to a study of this complex hydrolytic system and the role of the apo- protein cofactors.