Purification and Properties of Brain Cathepsin B

Abstract

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...

Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

“…More recently, several workers have reported the presence of additional thiol endopeptidases in lysosomes-e.g., cathepsins T, N, P, and S (12-15)-but their amounts appear to be very low. These lysosomal thiol endopeptidases are considered to play an important role in intracellular protein degradation and possibly in post-translational processing of some biologically important substances such as endorphin (16) and insulin (14,17).…”

Distribution of Cystatin C (γ-Trace), an Inhibitor of Lysosomal Cysteine Proteinases, in the Anterior Lobe of Simian and Human Pituitary Glands

“…The cysteine proteinases are thought to be respon sible for a significant part of the intracellular protein turn over [4,15,23]. High cathepsin B-like activity with the ca pacity to hydrolyze [3-lipotropin and |3-endorphin has been found in the anterior lobe of the pituitary glands of pig and rat [6,28], and it may be that the occurrence of cystatin C in certain adenohypophyseal cells indicates a role of the pro tein in the regulating mechanism of the intracellular de gradation of proteins or in the processing of peptides from their precursors in these cells. The blurred and indistinct immunostaining of cystatin C in human tissue taken within a few hours of death ( fig.…”

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

“…Cathepsin B purified from calf brain has been shown to split B-lipotropin into several products (Suhar and Marks, 1979;Marks et al, 1980). The products and cleavage sites have not been determined, however.…”

“…Interestingly, an aminopeptidase purified from human brain removing the NH2-terminal tyrosine from fl-endorphin-(1-5) is unable to cleave the NH2-terminus of fl-endorphin-(1-16) (Traficante et al, 1980). Cathepsin B purified from bovine brain has been reported to cleave fl-endorphin (Suhar and Marks, 1979). However, the site of cleavage has not been determined as yet.…”