Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.
Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.
Baricos
W H
WH
Department of Biochemistry, Tulane Medical School, New Orleans, LA 70112.
Zhou
Y
Y
Mason
R W
RW
Barrett
A J
AJ
D001485:Basement Membrane / Q000201:enzymology; D002401:Cathepsin B / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases; D004151:Dipeptides; D010450:Endopeptidases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000201:enzymology*; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D003374:Coumarins; D004151:Dipeptides; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D002403:Cathepsins; D010450:Endopeptidases; D010196:Pancreatic Elastase; D014357:Trypsin; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D003012:Microbial Collagenase