Identification of the active site cysteine and of the disulfide bonds in the N‐terminal part of the molecule of bovine spleen cathepsin B

Pohl, Jan; Baudyš, Miroslav; Tomášek, V.; Kostka, V.

doi:10.1016/0014-5793(82)80210-x

Cited by 30 publications

(12 citation statements)

References 22 publications

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“…Alternatively, if within the lysosome all the cysteinyl residues existed in the reduced form, they could have become oxidized to disulfide bonds during the isolation procedures. Pohl et al (40) recently reported a disulfide bond in the light chain of bovine spleen cathepsin B although compelling evidence was not pre- sented. Their result agrees with one of our hypothetical disulfide bonds in rat liver cathepsin B (Cys-14-Cys-43).…”

Section: Resultsmentioning

confidence: 99%

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

Takio¹,

Towatari²,

Katunuma³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

206

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(20) and actinidin (21).The experimental details of the sequence analysis of cathepsins B and H will be published elsewhere. MATERIALS AND METHODSCrystalline rat liver cathepsin B was prepared as described (11). Cathepsin H was prepared by the method of Schwartz and Barrett (22). The intact chain and the two polypeptide chains of each enzyme, generated by proteolytic cleavage, were separated on a Sephacryl S-200 column after S-carboxymethylation of reduced disulfide bonds as reported (19). Trypsin treated with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK-trypsin) and Staphylococcus aureus V8 protease were obtained from Worthington and Miles, respectively. Gel filtration media were products of Pharmacia.Chemical cleavages by cyanogen bromide (Eastman) and by 2 -(2 -nitrophenylsulphenyl) -3-methyl -3'-bromoindolenine (BNPS-skatole) (Pierce) were carried out by methods of Gross and Witkop (23) (30,31).A search for a homologous sequence was carried out with a VAX/VMS computer by using a "protein sequence database" of the Atlas of Protein Sequence and Structure (version 4, April 30, 1982) obtained from the National Biomedical Research Abbreviations: TPCK-trypsin, trypsin treated with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone; BNPS-skatole, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine. 3666The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Resultsmentioning

confidence: 99%

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

Takio¹,

Towatari²,

Katunuma³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

206

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“…Cleavage of HPMA copolymer-drug conjugates by lysosomal enzymes in vitro Enzymes Two sources of lysosomal enzymes were used: a mixture of rat liver lysosomal enzymes (tritosomes) prepared according to the method of Trouet (1974) and two purified lysosomal enzymes isolated from bovine spleen (Pohl et al, 1982), cathepsin B (EC. 3.4.22.1) and cathepsin L (EC.…”

Section: Tyr-nh2 Pmentioning

confidence: 99%

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

Duncan¹,

Kopečkova-Rejmanová²,

Strohalm³

et al. 1987

Br J Cancer

166

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Summary During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-PheLeu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184,ugml-1) could be selected to achieve a cytotoxic effect.

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“…Our preliminary investigation of P. tenuis identified several distinct cathepsin B-like transcripts, a result that is compatible with multigenic organization or allelic diversification. Whereas PtCPR-2 bears all the features of an active protease, PtCPR-1 and other paralogous sequences lack the essential active-site cysteine for which this class of proteases is named (34). Such C29G cathepsin B mutants have not been described for other organisms.…”

Section: Discussionmentioning

confidence: 99%

Cathepsin B Homologue at the Interface between a Parasitic Nematode and Its Intermediate Host

et al. 2006

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Parelaphostrongylus tenuis is a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsis sp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence of P. tenuis from the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.

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Identification of the active site cysteine and of the disulfide bonds in the N‐terminal part of the molecule of bovine spleen cathepsin B

Cited by 30 publications

References 22 publications

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

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

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

Cathepsin B Homologue at the Interface between a Parasitic Nematode and Its Intermediate Host

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