Secretion-coupled increase in the catalytic activity of rat hepatic lipase.
Freshly isolated rat hepatocytes synthesize and secrete hepatic lipase (HL). Comparison of secreted HL with intracellular HL indicates a secretion-linked increase in the specific enzyme activity. (a) Immunotitration with polyclonal anti-HL showed a 3-5-fold lower specific enzyme activity of intracellular HL than of secreted HL. This was confirmed by ELISA using a mixture of monoclonal anti-HL's. (b) After isolation on Sepharose-heparin, a similar difference in specific enzyme activity was observed, whereas the apparent Km for glyceroltrioleate was not different. (c) HL activity secreted in the absence of protein de novo synthesis was 5-fold higher than was accounted for by the fall in intracellular activity, whereas HL protein lost from the cells was near-completely recovered in the extracellular medium. (d) The presence of inactive HL protein was demonstrated in cells treated with castanospermine, which inhibits secretion of newly synthesized HL by interfering with maturation at an early stage of N-linked oligosaccharide processing. Upon removal of castanospermine, secretion of HL activity recovered, even when protein de nove synthesis was inhibited, strongly suggesting that part of the inactive HL was mobilized and became activated. This secretion-coupled increase in HL activity in the absence of protein synthesis suggests the existence of inactive precursor within rat hepatocytes. The catalytic activity of HL becomes apparent upon maturation of the protein after oligosaccharide processing by the rough endoplasmic reticulum glucosidases.
Verhoeven
A J
AJ
Department of Biochemistry I, Erasmus University Rotterdam, The Netherlands.
Jansen
H
H
D000818:Animals; D002384:Catalysis; D002846:Chromatography, Affinity; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005959:Glucosidases / Q000037:antagonists & inhibitors; D007212:Indolizines / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D054836:Indolizidines
D003513:Cycloheximide; D005959:Glucosidases; D007212:Indolizines; D008049:Lipase; D011500:Protein Synthesis Inhibitors; C037806:castanospermine; D054836:Indolizidines