Comparison of the degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane in rat hepatocytes. Implications for the cytomorphological basis of protein catabolism.
The degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane has been compared in rat hepatocyte monolayers. Monoamine oxidase was specifically irreversibly radiolabelled by the suicide inhibitor [3H]pargyline. Hepatocyte monolayers were cultured in conditions in which rates of protein catabolism like those in vivo are maintained [Evans & Mayer (1983) Biochem. J. 216, 151-161]. Incubation of hepatocyte monolayers for 17 h with [3H]pargyline specifically radiolabels mitochondrial monoamine oxidase, as shown by Percoll-gradient fractionation of broken hepatocytes. Monoamine oxidase is degraded at a similar rate to that observed in liver in vivo (t1/2 approx. 63 h). The effects of leupeptin, methylamine and colchicine on the degradation of endogenous radiolabelled enzyme has been studied over prolonged culture periods. Culture of hepatocytes for periods of up to 80 h with inhibitors was not cytotoxic, as demonstrated by measurements of several intrinsic biochemical parameters. Leupeptin, methylamine and colchicine inhibit the degradation of endogenous monoamine oxidase by 60, 38 and 18% respectively. Monoamine oxidase in mitochondrial-outer-membrane vesicles introduced into hepatocytes by poly(ethylene glycol)-mediated vesicle-cell transplantation is degraded at a similar rate (t1/2 55 h) to the endogenous mitochondrial enzyme. Whereas leupeptin inhibits the degradation of endogenous and transplanted enzyme to a similar extent, methylamine and colchicine inhibit the degradation of transplanted enzyme to a much greater extent (85 and 56% respectively). Fluorescence microscopy (with fluorescein isothiocyanate-conjugated mitochondrial outer membrane) shows that transplanted mitochondrial outer membrane undergoes internalization and translocation to a sided perinuclear site, as observed previously with whole mitochondria [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The effects of the inhibitors on the distribution of transplanted membrane material in the cell and inhibition of proteolysis show the importance of cytomorphology for intracellular protein catabolism.
D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D007425:Intracellular Membranes / Q000201:enzymology / Q000637:transplantation; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000637:transplantation / Q000648:ultrastructure; D008995:Monoamine Oxidase / Q000378:metabolism*; D010293:Pargyline / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats
D011480:Protease Inhibitors; D010293:Pargyline; D008995:Monoamine Oxidase; D007930:Leucine