Intracellular activation of rat hepatic lipase requires transport to the Golgi compartment and is associated with a decrease in sedimentation velocity.
Hepatic lipase (HL) is an N-glycoprotein that acquires triglyceridase activity somewhere during maturation and secretion. To determine where and how HL becomes activated, the effect of drugs that interfere with maturation and intracellular transport of HL protein was studied using freshly isolated rat hepatocytes. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), castanospermine, monensin, and colchicin all inhibited secretion of HL without affecting its specific enzyme activity. The specific enzyme activity of intracellular HL was decreased by 25-50% upon incubation with CCCP or castanospermine, and increased 2-fold with monensin and colchicin. Glucose trimming of HL protein was not affected by CCCP, as indicated by digestion of immunoprecipitates with jack bean alpha-mannosidase. Pulse labeling experiments with [(35)S]methionine indicated that conversion of the 53-kDa precursor to the 58-kDa form, nor the development of endoglycosidase H-resistance, were essential for acquisition of enzyme activity. In sucrose gradients, HL protein from secretion media sedimented as a homogeneous band of about 5.8 S, whereas HL protein from the cell lysates migrated as a broad band extending from 5.8 S to more than 8 S. With both sources, HL activity was exclusively associated with the 5.8 S HL protein form. We conclude that glucose trimming of HL protein in the endoplasmic reticulum is not sufficient for activation; full activation occurs during or after transport from the endoplasmic reticulum to the Golgi and is associated with a decrease in sedimentation velocity.
Verhoeven
A J
AJ
Department of Biochemistry, Cardiovascular Research Institute (COEUR), Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands. verhoeven@BC1.FGG.EUR.NL
Neve
B P
BP
Jansen
H
H
D000818:Animals; D001692:Biological Transport; D001704:Biopolymers; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D003078:Colchicine / Q000494:pharmacology; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000201:enzymology*; D007212:Indolizines / Q000494:pharmacology; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D054836:Indolizidines
D001704:Biopolymers; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003078:Colchicine; D007212:Indolizines; D008049:Lipase; D008985:Monensin; C037806:castanospermine; D054836:Indolizidines