Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.
Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.
Ohshita
T
T
Division of Enzyme Chemistry, University of Tokushima, Japan.
Nikawa
T
T
Towatari
T
T
Katunuma
N
N
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D002850:Chromatography, Gel; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D060748:Fetuins; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D060748:Fetuins; D005456:Fluorescent Dyes; D000509:alpha-Fetoproteins; C017029:asialofetuin; C068250:N-(3-propylcarbamoyloxirane-2-carbonyl)-isoleucyl-proline; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D002401:Cathepsin B; D007930:Leucine; D016650:Fluorescein-5-isothiocyanate