Human cathepsin B. Application of the substrate <i>N</i>-benzyloxycarbonyl-<scp>l</scp>-arginyl-<scp>l</scp>-arginine 2-naphthylamide to a study of the inhibition by leupeptin

Knight, Christopher G.

doi:10.1042/bj1890447

Cited by 89 publications

(26 citation statements)

References 28 publications

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“…Other investigators have dealt with the inhibition mechanism of cathepsin B by leupeptin. For human liver cathepsin B, Knight reported values around 7 nM when using a racemic mixture of two leupeptins (N-proionyl-leupeptin and N-acetyl-leupeptin in a ratio 3 : 1, respectively) in good agreement with the present results [32]. The remarkable effects of the N-terminal substituents on the inhibition constants of leupeptin analogs have been discussed by Borin et al [35].…”

Section: Pltrificalion and Znhihirion Qf' Cathepsin Bsupporting

confidence: 81%

“…This method allows the unequivocal classification of the inhibition type even in very critical cases. The inhibition type for leupeptin and cathepsin B was found to be fully competi- tive, confirming previous results [32]. Ki values obtained by these two methods were the same within the experimental error and are given in Table 2 as means from several (at least four) separate experiments in which the enzyme was preincubated with inhibitor standard deviations.…”

Section: Steady-state Inhibition O J Cuthrpsin B By Leupeptinsupporting

confidence: 74%

“…Confirming existing results obtained with human liver cathepsin B [32], the enzyme from human spleen and rabbit liver was also inhibited by leupeptin with a fully competitive mechanism. In addition to this, leupeptin behaved as a slow- Therefore, leupeptin can be classified as a slow, tight-binding inhibitor of cathepsin B [34].…”

Section: Pltrificalion and Znhihirion Qf' Cathepsin Bsupporting

confidence: 71%

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The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Baici¹,

1982

European Journal of Biochemistry

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Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k,, and k o f f for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio koff/kon, was in good agreement with the inhibition constant obtained using a steady-state treatment. k,, was 1.8 x lo5 M-' s p l and 2 . 0~ lo5 M-' s-' for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M -' S -' . The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 20 pM, at least. This contrasts with the predictions drawn from the value of K , ( z 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 pM.Leupeptins are peptide derivatives isolated from culture filtrates of some Strqitomjxvs species [I, 21 and identified as mixtures of N-u-acyl-tripeptides containing the unusual amino aldehyde arginal at the C-terminal position. Leupeptins are potent competitive inhibitors of cysteine proteinases such as cathepsin B and papain [1,3] and serine proteinases such as trypsin, plasmin, kallikrein and acrosin [4,5].Cathepsin B is a lysosomal proteinase containing an essential cysteine residue in the active center and is found in the tissues of many animal species 161. While the normal physiological function of cathepsin B may be the intracellular breakdown of proteins [7,8], an implication of this enzyme has been proposed also in some pathological situations as cartilage destruction 191, tumor invasion [lo, 1 I] and muscular dystrophy [I 21.Recent work in this laboratory on the role of extracellular proteinases in the processes of tissue destruction in rheumatoid arthritis 1131 and tumor invasion [11,14] led us to the study of the inhibition mechanisms of these enzymes by some agents of established therapeutic significance [I5 -171. We have undertaken the present investigation on the inhibition of cathepsin B by leupeptin in virro in order to gain more information on the properties of the system before attempting experiments in vivo.The present paper reports on: (a) the purification of human spleen cathepsin B ; (b) the theory of tight-binding inhibition as a necessary complement to that which is k...

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Section: Pltrificalion and Znhihirion Qf' Cathepsin Bsupporting

confidence: 81%

Section: Steady-state Inhibition O J Cuthrpsin B By Leupeptinsupporting

confidence: 74%

Section: Pltrificalion and Znhihirion Qf' Cathepsin Bsupporting

confidence: 71%

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The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Baici¹,

1982

European Journal of Biochemistry

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“…These peptide aldehydes are analogues of the cysteine/serine protease inhibitor leupeptin (acetylor propionyl-Leu-Leu-arginal), which was reported to inhibit, among others, cathepsin B (K i , 7 nM) [21] and cathepsin L (K i , 37 nM) [10]. Furthermore, several di-and tripeptidyl aldehyde inhibitors were synthesized and their inhibitory effects investigated toward cysteine proteases including cathepsins B and L [1].…”

Section: Resultsmentioning

confidence: 99%

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Itô

Watanabe

Kim

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC 50 ) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl-and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC 50 , 12 nM) and L (IC 50 , 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.

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“…Cathepsin B catalytic activity was measured on the peptide sequence carbobenzoxy-Larginyl-arginine (z-Arg-Arg), a specific substrate that does not cross-react with other cathepsins (Knight, 1980), linked to 7-amino-4-methyl-coumarin (z-R-R-AMC, Calbiochem, San Diego, CA, U.S.A.). Proteins (30 g) were diluted in 95 L of a reaction buffer composed of (in micromoles per liter) sodium acetate 400 (pH 5.5) and L-cysteine 8.…”

Section: Cathepsin B Activity Assaymentioning

confidence: 99%

Activation of Proinflammatory Caspases by Cathepsin B in Focal Cerebral Ischemia

Benchoua

Braudeau

Reis

et al. 2004

J Cereb Blood Flow Metab

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Summary: Cathepsins and caspases are two families of proteases that play pivotal roles in ischemic cell death. This study investigated the existence of a cross-talk between cathepsin B and proinflammatory caspases in stroke-induced cell death, as recently suggested by in vitro data. Cortical ischemic damage was induced in mice by distal and permanent occlusion of the middle cerebral artery. Cytoplasmic activation of cathepsin B was observed from the early stages of infarction, and displayed an activation pattern parallel to the activation pattern of caspase-1 and -11. Immunohistochemistry revealed the colocalization of cathepsin B with each caspase in cells of the infarct core. The apical position of cathepsin B in both caspase-activation cascades was confirmed by pretreatment of the animals with the cathepsin B inhibitor CA-074, which also potently protected cortical structures from ischemic damage, indicating involvement of the proteases in the lesion process. The results show that cathepsin B release is an early event following occlusion of cerebral arteries, which eventually triggers the activation of proinflammatory caspases in the absence of reperfusion. This new pathway may play a critical role in brain infarction by promoting inflammatory responses, and/or by amplifying the apoptotic process.

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Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide to a study of the inhibition by leupeptin

Cited by 89 publications

References 28 publications

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

The Slow, Tight‐Binding Inhibition of Cathepsin B by Leupeptin

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Activation of Proinflammatory Caspases by Cathepsin B in Focal Cerebral Ischemia

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