Purification and Properties of Brain Cathepsin B

Abstract

CM‐cellulose chromatography of calf brain extracts revealed the presence of several enzymes hydrolysing benzoyiarginyl‐β‐naphthylamide (Bz‐Arg‐Nap) one of which resembled cathepsin B (EC 3.4.22.1). The latter was purified to homogeneity and showed an absolute dependence on thiol compounds and was optimally active at pH 6.5. The molecular weight of the purified enzyme was 27000 ± 1500. Purified enzyme was inhibited by leupeptin, antipain, iodoacetate, Hg 2+ , zn 2+ , and partially by chymostatin and phenylmethylsulfonyl fluoride but was unaffected by pepstatin, puromycin, bestatin and bacitracin. The K m with Bz‐Arg‐Nap was 0.53 mM, K cat 4.5 min −1 . Dipeptidyl and tripeptidyl‐acrylamide substrates were hydrolysed also by the brain enzyme but with lower K cat / K m ratios. Comparison of the kinetics of inhibition using leupeptin (Ac‐ l ‐Leu‐Leu‐arginal) with Boc‐ d ‐Phe‐Pro‐arginal showed that the latter was non‐competitive and the former competitive with K i of 1.5*10 −8 M and 8*10 −7 M respectively. Purified enzyme hydrolysed histones (lysine‐rich and type‐1), myelin basic protein, porcine β‐lipotropin, human β‐endorphin and protamine. Hydrolysis of β‐lipotropin was accompanied by the generation of a 8000‐ M r fragment. Cathepsin‐B‐like enzymes were present in highest concentration in rat pituitary, and lower in cerebellum, hypothalamus, medulla oblongata, striatum and spinal cord.