title: Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes.
abstract: Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.
mesh_terms: D000818:Animals; D006026:Glycoside Hydrolases / Q000378:metabolism; D007158:Immunologic Techniques; D008049:Lipase / Q000096:biosynthesis*; D008099:Liver / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D014415:Tunicamycin; D008715:Methionine; D008049:Lipase; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase