Abstract
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.
Highlights
The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552
TG lipolysis is under hormonal regulation by the neuropeptide adipokinetic hormone (AKH) [9], which elicits a glucagon-like action mediated by a G protein-coupled receptor that activates both inositol phosphate and cAMP signaling responses [10, 11]
The sequence VEVLPIS is found in CG8552-phosphatidic acid (PA), whereas the other two peptides shared a high degree of identity with two regions present in CG8552-PA (Fig. 1)
Summary
The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. The lipolytic response induced by AKH is associated with a rapid activation of fat body cAMPdependent protein kinase A (PKA) and a sustained increase in calcium influx [13]. The major TG-lipase of the fat body has been purified from adult M. sexta [14] This is a cytosolic enzyme with a molecular mass of 76 kDa that can be phosphorylated in vitro by PKA. Vokes a rapid phosphorylation of Lsdp, a protein that shares a small region of sequence identity with perilipin A from mammalian lipid droplets [18], and this event accounts for the majority of the lipolytic response induced by AKH [16, 17]
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