Specific spectrophotometric assays for cathepsin B1

Bajkowski, Andrew S.; Frankfater, Allen

doi:10.1016/0003-2697(75)90685-5

Cited by 58 publications

(33 citation statements)

References 16 publications

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“…To assess the activity of thiol proteases directly, CLN was used ( 18) as a specific substrate for cathepsin B, a thiol protease. Cathepsin B, along with cathepsin D, an aspartic protease, was shown to be a principal enzyme responsible for lysosomal digestion of LDL (6).…”

Section: Resultsmentioning

confidence: 99%

“…The degradation of the radioactive substrate was measured as TCA-soluble, noniodine radioactivity. Cathepsin B activity of macrophage extracts was assayed as follows: 0.5 og of extract was mixed with 500 81I of acetate buffer, pH 4.0, containing CLN at a final concentration of 2 x 10' M and absorbance at 326 nm monitored over a 30-min time interval (18). To determine the proteolytic activity of enzymes that have been stimulated by the addition of DTT, we conducted all experiments in the presence and absence of DTT.…”

Section: Methodsmentioning

confidence: 99%

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Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

1994

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

1994

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“…1). It is therefore not surprising that widely varying pH-activity profiles have been reported in the literature for different substrates (Knight, 1980;Bajkowski & Frankfater, 1983;Katunuma et al, 1983;Hirao et al, 1984;Willenbrock & Brocklehurst, 1984). The most striking differences between pH-activity profiles of cathepsin B and papain is the presence of a group with a PKa of about 5.4 modulating the activity of the former enzyme towards substrates bearing an arginine residue in P2.…”

Section: Kinetic Measurementsmentioning

confidence: 99%

“…On the basis of this result and the observed increase of activity towards reactivity probes by a deprotonation with a pKa of 3.4, it has been proposed that formation of the catalytic thiolateimidazolium ion-pair, which is believed to be the active form of papain and cathepsin B, is a necessary but not sufficient condition for catalysis by cathepsin B (Willenbrock & Brocklehurst, 1984. Cathepsin B, however, is active at low pH towards other substrates (Bajkowski & Frankfater, 1983;Katunuma et al, 1983;Hirao et al, 1984) and ionization of the group with a pKa of about 5.4 is observed only when an arginine residue is present at the P2 position of the substrate under study.We have used a systematic approach to characterize the S2 binding-site specificity of cathepsin B, based on three synthetic substrates of the enzyme. These substrates are of the type CbzXaa-Arg-NH-Mec, where Xaa represents L-phenylalanine, Larginine or L-citrulline.…”

mentioning

confidence: 99%

A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses

Khouri

Plouffe

Hasnain

et al. 1991

Biochemical Journal

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1. Three synthetic substrates of cathepsin B (EC 3.4.22.1) with various amino acid residues at the P2 position (Cbz-PheArg-NH-Mec, Cbz-Arg-Arg-NH-Mec and Cbz-Cit-Arg-NH-Mec, where Cbz represents benzyloxycarbonyl and NH-Mec represents 4-methylcoumarin-7-ylamide) were used to investigate the pH-dependency of cathepsin B-catalysed hydrolyses and to obtain information on the nature of enzyme-substrate interactions. 2. Non-linear-regression analysis of pH-activity profiles for these substrates indicates that at least four ionizable groups on cathepsin B with pKa values of 3.3, 4.55, 5.46 and > 7.3 can affect the rate of substrate hydrolysis. 3. Ionization of the residue with a pKa of 5.46 has a strong effect on activity towards the substrate with an arginine in P2 (8.4-fold increase in activity) but has only a moderate effect on the rate of hydrolysis with Cbz-Cit-Arg-NH-Mec (2.3-fold increase in activity) and virtually no effect with Cbz-Phe-Arg-NH-Mec. The kinetic data are consistent with this group being an acid residue with a side chain able to interact with the side chains of an arginine or a citrulline in the P2 position of a substrate. Amino acid sequence alignment and model building with the related enzyme papain (EC 3.4.22.2) INTRODUCTIONCathepsin B (EC 3.4.22.1) is a lysosomal cysteine proteinase that has been isolated from many mammalian tissues, including human liver (Barrett, 1977). Much of the information on the mechanism of cathepsin B has been obtained through comparisons with the well characterized cysteine proteinase papain (EC 3.4.22.2). Alignment and comparison of the sequences of related cysteine proteinases suggest similar threedimensional structures for cathepsin B and papain (Kamphuis et al., 1985;Dufour, 1988). Even though the basic features of the mechanisms are similar, major differences exist between the two enzymes. Cathepsin B is both an endopeptidase and a dipeptidyl carboxydipeptidase, whereas papain works exclusively as an endopeptidase (Aronson & Barrett, 1978;Bond & Barrett, 1980;McKay et al., 1983;Mason, 1989). The specificities of the two enzymes are also somewhat different. Papain shows a marked preference for peptides with a bulky non-polar side chain, especially L-phenylalanine, on the N-terminal side of the amino acid containing the peptide bond being hydrolysed, i.e. at the P2 position (Berger & Schechter, 1970). This specificity is attributed to interaction of the phenylalanine side chain with hydrophobic residues at the S2 subsite of papain, mainly and (Drenth et al., 1976). (Cathepsin B numbering is used throughout the text except for residues of papain, where the position number for papain is followed in parentheses by the corresponding number for cathepsin B.) Cathepsin B activity towards substrates with a P2 phenylalanine residue is still very high, but the enzyme also has a strong affinity for substrates with an arginine residue at the P2 position, such as Cbz-Arg-Arg-NHNap and Cbz-Arg-Arg-NH-Mec (Barrett & Kirschke, 1981).Ionization of a group with a pKa of ab...

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“…The concentration of [ 3 H]STX-binding sites in samples of saxiphilin was assayed according to the method described (10). The protein concentration of papain and cathepsins B and L was determined by absorption measurements at 280 nm using the molar absorption coefficients of 56,200 (29), 44,000 (30), and 44,300 M Ϫ1 cm…”

Section: Sds-page and Glutaraldehyde Cross-linking-sds-page Was Perfomentioning

confidence: 99%

Saxiphilin, a Saxitoxin-binding Protein with Two Thyroglobulin Type 1 Domains, Is an Inhibitor of Papain-like Cysteine Proteinases

Lenarčič¹,

Krishnan²,

Borukhovich³

et al. 2000

Journal of Biological Chemistry

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Specific spectrophotometric assays for cathepsin B1

Cited by 58 publications

References 16 publications

Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses

Saxiphilin, a Saxitoxin-binding Protein with Two Thyroglobulin Type 1 Domains, Is an Inhibitor of Papain-like Cysteine Proteinases

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