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Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.
Biochem J. 1994 Aug 01; 301 (Pt 3):727-35.BJ

Abstract

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Authors+Show Affiliations

Genzyme Corporation, Cambridge, MA 02139.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

8053898

Citation

Napper, A D., et al. "Purification and Characterization of Multiple Forms of the Pineapple-stem-derived Cysteine Proteinases Ananain and Comosain." The Biochemical Journal, vol. 301 (Pt 3), 1994, pp. 727-35.
Napper AD, Bennett SP, Borowski M, et al. Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain. Biochem J. 1994;301 (Pt 3):727-35.
Napper, A. D., Bennett, S. P., Borowski, M., Holdridge, M. B., Leonard, M. J., Rogers, E. E., Duan, Y., Laursen, R. A., Reinhold, B., & Shames, S. L. (1994). Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain. The Biochemical Journal, 301 (Pt 3), 727-35.
Napper AD, et al. Purification and Characterization of Multiple Forms of the Pineapple-stem-derived Cysteine Proteinases Ananain and Comosain. Biochem J. 1994 Aug 1;301 (Pt 3):727-35. PubMed PMID: 8053898.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain. AU - Napper,A D, AU - Bennett,S P, AU - Borowski,M, AU - Holdridge,M B, AU - Leonard,M J, AU - Rogers,E E, AU - Duan,Y, AU - Laursen,R A, AU - Reinhold,B, AU - Shames,S L, PY - 1994/8/1/pubmed PY - 1994/8/1/medline PY - 1994/8/1/entrez SP - 727 EP - 35 JF - The Biochemical journal JO - Biochem. J. VL - 301 (Pt 3) N2 - A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/8053898/Purification_and_characterization_of_multiple_forms_of_the_pineapple_stem_derived_cysteine_proteinases_ananain_and_comosain_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/bj3010727 DB - PRIME DP - Unbound Medicine ER -