The main triglyceride-lipase from the insect fat body is an active phospholipase A(1): identification and characterization.
The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.
Arrese
Estela L
EL
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA. estela@biochem.okstate.edu
Patel
Rajesh T
RT
Soulages
Jose L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005216:Fat Body / Q000201:enzymology*; D007301:Insect Hormones / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008066:Lipolysis; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007301:Insect Hormones; D019476:Insect Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C044833:adipokinetic hormone; D008049:Lipase; D010741:Phospholipases A; D011761:Pyrrolidonecarboxylic Acid