Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase.
A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.
Ohshita
Takeyuki
T
Department of Human Health and Nutrition, Faculty of Comprehensive Human Sciences, Shokei Gakuin College, Natorisi, Miyagi, Japan. ohshita@shokei.ac.jp
Hiroi
Yuzo
Y
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D015891:Cystatins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D007770:L-Lactate Dehydrogenase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; D007930:Leucine; C032854:leupeptin