Heterogeneity of Triglyceride Lipase of Rat Adipocytes

1 Triglyceride and monoglyceride lipase activities were compared in different preparations from rat adipose tissue or isolated adipocytes: fat‐free homogenate, 105000 × g fat‐free infranatant (soluble fraction) and 105000 × g sediment (particulate fraction) were assayed. The ratio of monoolein to triolein cleavage was 6.8 in whole homogenate and 1.9 and 20 in particulate and soluble fractions, respectively. Lipoprotein lipase activity of these enzyme preparations was not measurable under the experimental conditions used. 2 Since specific activities especially of triglyceride lipase(s) were low, other substrates were tested to increase the sensitivity. Among them 2‐naphthol laurate was found to be hydrolyzed by the whole homogenate at a rate about 50 times higher than triolein. 3 2‐Naphthol laurate was readily hydrolyzed by lipases in all preparations studied. Dioxane and ethylene glycol, the typical inhibitors of monoglyceride lipase activity strongly inhibited its cleavage in the soluble fraction whereas the typical inhibitors of triglyceride lipase activity (Triton ×‐100, lanthanum nitrate, 2‐propanol) were effective in inhibiting 2‐naphthol laurate cleavage particularly in 105000 × g sediment. 4 Heat inactivation, pH optima and starch‐block electrophoresis confirmed the difference between lipases in particulate and soluble fractions. Whereas in the soluble fraction these criteria did not reveal further heterogeneity, the particulate fraction was apparently heterogenous and exhibited two pH optima at 6.5 and 8, a non‐linear rate of heat inactivation with time and two electrophoretic fractions, thus suggesting the presence of two distinct triglyceride lipases. 5 The monoglyceride lipase character of the soluble lipase is supported by the kinetic evidence of competition between monoolein and 2‐naphthol laurate. The particulate lipases, on the other hand, exhibited competitive inhibition of 2‐naphthol laurate cleavage by triolein, which supports their triglyceride lipase character. 6 2‐Naphthol laurate might be thus a valuable tool in the study of both monoglyceride and triglyceride lipase activities in adipose tissue. The possibility of high dilution of enzyme preparation excludes the interference of endogenous substrate, the preparation of a stable substrate emulsion is easy and the estimation of the first product of the reaction is rapid and simple.