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PMID: 7034724 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.

The Biochemical journal ·Vol. 197 ·No. 3 ·1981-09-01 ·Pages 739-46

Brocklehurst K, Baines BS, Malthouse JP

Abstract

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.

MeSH Terms

4-Chloro-7-nitrobenzofurazan Benzoxazoles Binding Sites Catalysis Chromatography, Affinity Cysteine Endopeptidases Disulfides Endopeptidases/isolation & purification Ficain Fluorescent Dyes Isomerism Kinetics Papain Pyridines Sulfhydryl Reagents

Chemicals

Benzoxazoles Disulfides Fluorescent Dyes Pyridines Sulfhydryl Reagents 1,4-(N-2-aminoethyl-2'-pyridyl disulfide)-7-nitrobenzo-2-oxa-1,3-diazole n-propyl 2-pyridyl disulfide Endopeptidases Cysteine Endopeptidases actinidain Papain Ficain 4-Chloro-7-nitrobenzofurazan

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Brocklehurst K

Baines B S

Malthouse J P

References (25)

25 references, click to expand

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Article Info

Journal

The Biochemical journal

Abbr.

Biochem J

ISSN

0264-6021

DOI

10.1042/bj1970739

Published

1981-09-01

Pages

739-46

Language

English

Region

England

NLM ID

2984726R

PMCID

PMC1163189

Subset

External Links

PubMed source DOI: 10.1042/bj1970739 PMC Full Text View journal details

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Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.

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