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 μM, 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 μM), the latter enzyme being sensitive to puromycin and benzethonium chloride as well. Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2 are potent irreversible inhibitors of brain cathepsin L with K2nd 150 000 and 137 000 M−1 s−1 respectively. Properties of the enzymes from human and bovine brain are similar.