Degradation of apolipoprotein B of low density lipoprotein by cultured bovine smooth muscle cells. Accumulation of intermediates in the presence of protease inhibitors.
The incubation of bovine aortic smooth muscle cells with 125I-labeled low density lipoprotein in the presence of protease inhibitors resulted in the significant intracellular accumulation of intact apolipoprotein B, as well as a number of high molecular weight degradation intermediates. This effect was brought about both by leupeptin, a specific inhibitor of thiol proteases (40% inhibition of degradation), as well as by the more general lysosomotrophic inhibitors, chloroquine and NH4Cl. Qualitatively identical spectra of degradation intermediates were formed in the presence of chloroquine and NH4Cl as determined by autoradiography of SDS-polyacrylamide gel electrophoretic fractions, ranging from the apolipoprotein B band (Mr = 340,000) to bands with molecular weights of less than 14,000. The effect of NH4Cl was reversible and release of inhibition resulted in the sequential loss of intermediates from the cells; those species having higher molecular weights disappearing first. Inhibition by leupeptin was associated with the accumulation of degradation products in the lower molecular weight range only (Mr less than or equal to 72,000). These results provide evidence that apolipoprotein B proteolysis progresses in distinct stages via specific breakdown products and suggest that the thiol cathepsins become more active later in the degradation pathway.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism*; D001055:Apolipoproteins B; D002417:Cattle; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*
D001053:Apolipoproteins; D001055:Apolipoproteins B; D007976:Leupeptins; D008077:Lipoproteins, LDL; D011480:Protease Inhibitors; D000643:Ammonium Chloride; D002738:Chloroquine