Chymopapain. Chromatographic purification and immunological characterization

Buttle, David J.; Barrett, Alan J.

doi:10.1042/bj2230081

Cited by 48 publications

(26 citation statements)

References 19 publications

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“…The different reactivities towards 2PDS exhibited by various chymopapain preparations have been disputed 19 ' 101 . Also, evidence has been given which suggests that the multiple Chromatographie forms of chymopapain have to be attributed to artifacts resulting from the commercial processing of thelatex [6] . Papaya proteinase Ω is the third proteolytic constituent of the papaya latex, being characterized by its extreme basicity with an isoelectric point higher than 11.0 Ι8 · 11 · 12] .…”

Section: Die Thiol-proteinasen Aus Dem Milchsaft Von Carica Papaya Lmentioning

confidence: 96%

“…The preparation at this stage of the purification was shown to be electrophoretically homogeneous and to contain 0.95 thiol function per enzyme molecule (homogeneity was firmly established in the course of amino-acid sequencing 1261 . M r (x 10~3) 6 1 8 The specific amidase activity which was 1.32 nkat/mg reached after affinity chromatography the value of 1.98 nkat/mg.…”

Section: ) Amino-acid Analysismentioning

confidence: 97%

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The Thiol Proteinases from the Latex ofCarica papayaL. I. Fractionation, Purification and Preliminary Characterization

Dubois

Jacquet²,

Schnek³

et al. 1988

Biological Chemistry Hoppe-Seyler

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Three thiol proteinases, namely papain, chymopapain and proteinase Ω were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as iS-sulphenylthiosulfate derivative or after blocking with pchloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase Ω. The active sites of chymopapain and proteinase Ω could not be distinguished from that of papain neither by the analysis of the pH dependence of Κςχ/Κη nor by the examination of the pH dependence of the fluorescence emission spectra. Die Thiol-Proteinasen aus dem Milchsaft vonCarica papaya L. I. Fraktionierung, Reinigung und vorl ufige Charakterisierung Zusammenfassung: Aus dem Milchsaft des Melonenbaumes (Carica papaya L.) wurden die drei Thiol-Proteinasen Papain, Chymopapain und Proteinase Ω isoliert und bis zur Einheitlichkeit gereinigt. W hrend der Aufarbeitung wurde die ThiolFunktion der Cysteinreste gesch tzt, und zwar durch Umwandlung in gemischte Disulfide mittels Cysteamin, 2-Thiopyridon, S-Sulphenylthiosulfat-Derivate oder durch Umsetzung mit p-Chloromercuribenzoes ure. Im Gegensatz zu fr heren Ver ffentlichungen erwies sich Chymopapain ebenfalls als Monothiol-Proteinase wie Papain und Proteinase Ω. Die aktiven Zentren von Chymopapain und Proteinase Ω konnten weder durch die pHAbh ngigkeit der kau/Km -Werte noch durch ihre Fluoreszenz-Emissionsspektren vom aktiven Zentrum des Papains unterschieden werden.

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Section: Die Thiol-proteinasen Aus Dem Milchsaft Von Carica Papaya Lmentioning

confidence: 96%

Section: ) Amino-acid Analysismentioning

confidence: 97%

The Thiol Proteinases from the Latex ofCarica papayaL. I. Fractionation, Purification and Preliminary Characterization

Dubois

Jacquet²,

Schnek³

et al. 1988

Biological Chemistry Hoppe-Seyler

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“…These were prepared and assayed as described in the references cited: cathepsin H (Schwartz & Barrett, 1980), cathepsin L (Mason et al, 1985), chymopapain and papain (Buttle & Barrett, 1984) and actinidin (Brocklehurst et al, 1981).…”

Section: Other Proteinasesmentioning

confidence: 99%

Purification of cathepsin B by a new form of affinity chromatography

Rich¹,

Brown²,

Barrett³

1986

Biochemical Journal

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Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

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“…Cysteine proteinases [1][2][3] have recently attracted renewed interest as they have been implicated in the progression of several human diseases [4,5]. One of the key challenges faced by workers studying cysteine proteinases, especially those of plant origin, has been the characterization of multiple enzyme forms, such as those found in preparations of chymopapain [6][7][8][9][10][11], ficin [12], stem bromelain [13,14] and the mammalian cathepsins [15]. Recently, the isolation of fully active enzymes from mixtures containing inactivated (usually oxidized) material has been facilitated by the use of either thiol-specific affinity chromatography [16,17] or covalent chromatography [18].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain

Napper¹,

Bennett²,

Borowski³

et al. 1994

Biochemical Journal

108

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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.

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Chymopapain. Chromatographic purification and immunological characterization

Cited by 48 publications

References 19 publications

The Thiol Proteinases from the Latex ofCarica papayaL. I. Fractionation, Purification and Preliminary Characterization

The Thiol Proteinases from the Latex ofCarica papayaL. I. Fractionation, Purification and Preliminary Characterization

Purification of cathepsin B by a new form of affinity chromatography

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain

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