3 . CM-cellulose chromatography of calf brain extracts revealed the presence of several enzymes hydrolysing benzoylarginyl-P-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 i 1500.2. Purified enzyme was inhibited by leupeptin, antipain, iodoacetate, Hg2+, Zn2+, and partially by chymostatin and phenylmethylsulfonyl fluoride but was unaffected by pepstatin, puromycin. bestatin and bacitracin. The Km with Bz-Arg-Nap was 0.53 mM, Kcat 4.5 min-l. Dipeptidyl and tripeptidyl-acrylamide substrates were hydrolysed also by the brain enzyme but with lower Kc,,/ K, ratios.3. Comparison of the kinetics of inhibition using leupeptin (Ac-L-Leu-Leu-arginal) with Boc-1,-Phe-Pro-arginal showed that the latter was non-competitive and the former competitive with Ki of 1.5 x l o p 8 M and 8 x 4. Purified enzyme hydrolysed histones (lysine-rich and type-1), myelin basic protein, porcine P-lipotropin, human P-endorphin and protamine. Hydrolysis of P-lipotropin was accompanied by the generation of a 800O-M, fragment.5. Cathepsin-B-like enzymes were present in highest concentration in rat pituitary, and lower in cerebellum, hypothalamus, medulla oblongata, striatum and spinal cord.
M respectively.Lysosomal cathepsin B, defined as the enzyme hydrolyzing benzoylarginyl-P-naphthylamide (BzArg-Nap) or its congeners, is considered to play a key role in intracellular protein turnover [l -31. Few studies have been done on this enzyme in brain even through this non-mitotic tissue is subject to protein renewal, and consequently requires breakdown for cellular homeostasis [4]. Interest in brain cathepsin stems also from its possible role in pathological processes such as demyelination [5], and in the processing of neuropeptides from precursors [6,7]. A role in physiological and pathological events is implied by the demonstration that purified cathepsin €3 from other sources can inactivate glycolytic enzymes [S, 91, release albumin from proalbumin [lo], or be involved in the processing of proinsulin [ l l ] and that tissue levels are altered in muscular dystrophy [12] and in malignancy ~3 1 .Ahhrevicition.r. Nap, 8-naphthylamide; PhMeS03F, phenylmethylsulfonyl fluoride; Boc, r-butoxycarbonyl ; Cbz, carbobenzoxy: Me2S0. dimethylsulfoxide; Bz, benzoyl./;/izjmw,\. CathepsinB (EC 3.4.22.1); cathepsinD (EC 3.4.23.5).Recent data suggest that tissues contain more than one enzyme that hydrolyzes Bz-Arg-Nap which can be differentiated by the use of synthetic and native substrates and by the effects of known inhibitors [14,15]. In previous studies Grynbaum and Marks (161 showed that brain enzymes hydrolyzing Bz-ArgNap in the presence of thiol compounds at pH 6.0 were localized in lysosomal enriched fractions. In the present study we report on a method capable of yielding a homogeneous preparation of calf brain cathepsin B, which is distinct from tha...
