Human and bovine brain cathepsin L and cathepsin H: purification, physico-chemical properties, and specificity.
Cathespin L (EC 3.4.22.15) and cathepsin H (EC 3.4.22.16) have been purified from brain cortex to apparent homogeneity by a simultaneous procedure involving acid extraction of homogenate at pH 4.2, ammonium sulfate fractionation (30-80%), chromatography on pepstatin-Sepharose, CM-Sephadex C-50, DEAE-Sephadex A-50, phenyl- and concanavalin A-Sepharose and isoelectric focusing. Cathepsin L and cathepsin H were assayed in the presence of dithiothreitol and Na2EDTA (2 mM each) with Z-Phe-Arg-NHMec (pH 5.5) and Lys-NNa (pH 6.5) respectively. Cathepsin L consists of 2 polypeptide chains with Mr 25,000 and 5,000, Mr of cathepsin H is 28,000. Cathepsin L exists in brain tissue in two multiple forms with pI values 5.7 and 5.9, pI of cathepsin H is 6.8. Substrate specificity of these thiol proteinases was tested with proteins (pyridoxyl-hemoglobin, azocasein) and low Mr naphthylamide and methylcoumarylamide substrates: Lys-NNa, Arg-NNa, Dz-Arg-NNa, Z-Arg-Arg-NNaOMe, Z-Phe-Arg-NHMec, Z-Phe, Val-Arg-NHMec, Z-Gly-Gly-Arg-NHMec. Z-Phe-Arg-NHMec is the best substrate for cathepsin L (KM = 5 microM, Kcat = 21 s-1), Arg-NNa--for cathepsin H (KM = 0.1 mM, Kcat = 1.93 s-1), being endoaminopeptidase cathepsin H also hydrolyses Bz-Arg-NNa (KM = 0.7 mM, Kcat = 1.3 s-1). Both proteinases are inhibited by traditional inhibitors of cysteine proteinases and E-64, but leupeptin turned to be more effective inhibitor of cathepsin L (Ki = 2.4 nM) than of cathepsin H (Ki = 9.2 microM), the latter enzyme being sensitive to puromycin and benzethonium chloride as well.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D056655:Cathepsin H; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D008970:Molecular Weight; D013379:Substrate Specificity
D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534126:CTSH protein, human; D056655:Cathepsin H