Abstract
Trioleinase activity of human adipose tissue was examined with special attention given to artifacts able to unvalidate the enzymatic assays. The results are partly a correction of data from a previous work (Boyer, J., Le Petit, J. and Giudicelli, H. (1970) Biochim. Biophys. Acta 210, 411–419), where fatty monoesters acting as substrates strongly altered the specificity of the triglyceride lipase assay. The yield of extraction of trioleinase from adipose tissue was improved by incubating the crude preparation at 50°C for 15 min, under glycerol protection. After a 35-fold purification of the enzyme, 16% of the total activity was found to be associated with material of low density collected after serial (NH 4) 2SO 4 fractionation of a pH-5.4-precipitated fraction. This lipid-bound lipase showed a sharp maximum of activity at pH 7.4 and an apparent K m towards triolein of 0.5 mM. The enzyme did not tolerate bile salts. It was neither stimulated by heparin or fresh human serum nor inhibited by 1 M NaCl, all properties consonant with those of “hormone-sensitive” lipase. None of the enzymatic preparations tested was found to contain an activity behaving differently from hormone-sensitive lipase and identifiable as lipoprotein lipase. It was shown that water-insoluble components present in the adipose tissue extracts appear to be readily adsorbed onto the substrate interface and may impair the process of formation of the enzyme-substrate complex. The measured levels of activity may therefore fluctuate in an unpredictable manner when lipase is assayed in systems of small volume, containing low concentrations of substrate and where the enzyme is introduced with variable amounts of tissue material. Trioleinase exhibited a maximal activity towards an emulsion of albuminbound triolein. Besides its role of fatty acid acceptor, albumin appeared to modify the substrate interface and to increase its susceptibility to the lipase action.
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