Purification and properties of a proline iminopeptidase from apricot seeds

A proline iminopeptidase was purified about 18,000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purif...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1982-01, Vol.92 (2), p.413-421
Main Authors: Ninomiya, K, Kawatani, K, Tanaka, S, Kawata, S, Makisumi, S
Format: Article
Language:English
Subjects:
Aminopeptidases - isolation & purification
Aminopeptidases - metabolism
Isoelectric Focusing
Kinetics
Molecular Weight
Oxidation-Reduction
Photochemistry
proline iminopeptidase
Prunus armeniaca
purification
seeds
Seeds - enzymology
Space life sciences
Substrate Specificity
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Summary:A proline iminopeptidase was purified about 18,000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purified enzyme had a molecular weight of 220,000 by gel filtration and 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the native enzyme may be composed of four identical subunits. The isoelectric point was 6.2 as determined by gel electrofocusing. The pH optimum for L-proline β-naphthylamide was between pH 7.5 and 8.0, and the enzyme was stable in the pH 6.5-to-8.0 region and up to 40°C. The enzyme was specific for L-proline β-naphthylamide among various amino acid β-naphthylamides, and it also hydrolyzed L-prolylglycine and L-prolylglycylglycine. The enzyme was strongly inhibited by p-chloromercuribenzoate, 5, 5′-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and heavy metal ions, but was not activated significantly by thiol compounds. Moreover, the enzyme was inactivated by diethyl pyrocarbonate, p-bromophenacyl bromide, and photooxidation, but was not affected by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bestatin, puromycin, or metal chelating agents. No activation of the enzyme was observed on addition of metal ions. These results suggest that the enzyme is not classifiable as a metalloenzyme, and that cysteine and histidine residues may participate in the enzyme activity.
ISSN:0021-924X
1756-2651
DOI:10.1093/oxfordjournals.jbchem.a133948