On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1

McDonald, J. Ken; Ellis, Stanley

doi:10.1016/0024-3205(75)90137-x

Cited by 170 publications

(37 citation statements)

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“…1) was considered to represent a homogeneous form of cathepsin B based on gel electrophoresis showing a single band o f protein associated with enzyme activity, the sharp pH optima and its other properties. These include a dependence on thiol compounds for activity, inhibition by leupeptin, iodacetate, heavy metal, and the ability to hydrolyze a number of other arylamide substrates such as Arg-Arg-Nap and Lys-Lys-Nap [14,31]. The M , of approximately 27500 is similar to that reported for other tissues but lower than that of rat brain enzyme partially purified by Distelmaier et al [32].…”

Section: Discussionsupporting

confidence: 60%

Purification and Properties of Brain Cathepsin B

Suhar

Marks

1979

European Journal of Biochemistry

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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...

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Section: Discussionsupporting

confidence: 60%

Purification and Properties of Brain Cathepsin B

Suhar

Marks

1979

European Journal of Biochemistry

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“…Potentially, a diversity of hydrolases present in saliva can be responsible for this cleavage. Cathepsins, for instance, are known to recognize and cleave a wide range of peptide bonds, among which are Arg-Arg, Lys-Lys, and Phe-Arg (6,10,15,22,30,32,42). As well as the cathepsins, other salivary proteases, like alanine aminopeptidase and dipeptidyl peptidase IV, may play a role in the cleavage of these three substrates (2).…”

Section: Discussionmentioning

confidence: 99%

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

et al. 2012

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Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain D-amino acids, led to the discovery of five P. gingivalisspecific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and L-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10 5 CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of L-amino acids and Bz-L-Arg-NHPhNO 2 showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single D-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of D-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

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“…Cathepsin B activity was measured fluorimetrically using CBZ-arginyl-arginine-B-napthylamide as the substrate as described by McDonald and Ellis (13) with minor modifications (4). Amylase, DNA, RNA, and cytochrome oxidase were evaluated as described previously (10).…”

Section: Methodsmentioning

confidence: 99%