Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta.
A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.
Arrese
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Department of Biochemistry, University of Arizona, Tucson 85721.
Wells
M A
MA
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004075:Diglycerides; D005216:Fat Body / Q000201:enzymology*; D005260:Female; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D018402:Manduca / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D013379:Substrate Specificity; D014280:Triglycerides
D004075:Diglycerides; D014280:Triglycerides; D007531:Isoflurophate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008049:Lipase; D008274:Magnesium