Purification and characterization of collagenolytic property of renal cathepsin L from arthritic rat.
1. This paper describes the purification and characterization of collagenolytic property of renal cathepsin L isolated from kidney of rats rendered adjuvant arthritis. The enzyme was isolated by acid extraction, ammonium sulfate fractionation, Sephadex gel filtration, CM-Sephadex chromatography and Sephacryl S-300 chromatography. 2. The enzyme preparation was found to be homogeneous by gel filtration and SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 29,000. 3. Incubation of rat tail tendon collagen with purified cathepsin L resulted a conversion of cross-linked beta-chain dimers into uncross-linked alpha-chain monomers. The pH optimum for collagen degradation by purified cathepsin L was found to be 3.5. This optimal pH is shifted to 4.5 when haemoglobin was used as a substrate for the enzyme. 4. Various activators and inhibitors were tested for their influence on the activity of cathepsin L. The purified enzyme showed a maximal activity in the presence of EDTA. Cysteine was also found to increase the activity of cathepsin L. This enzyme was strongly inhibited by iodoacetate, p-chloromercurobenzoate, mercuric chloride but not inhibited by pepstatin or PMSF. E-64 and leupeptin were also found to be strong inhibitors for cathepsin L. The degradation of rat tail tendon collagen by cathepsin L was completely inhibited by E-64. 5. The results presented in this investigation suggest that cathepsin L play a crucial role in the pathogenesis of adjuvant arthritis.
Reddy
G K
GK
Department of Biochemistry, Central Leather Research Institute, Adyar, Madras, India.
Dhar
S C
SC
D000818:Animals; D001169:Arthritis, Experimental / Q000201:enzymology* / Q000209:etiology; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D003094:Collagen; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat