The purification and properties of cathepsin L from rabbit liver.
Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D011817:Rabbits; D013379:Substrate Specificity
D000906:Antibodies; D007527:Isoenzymes; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L