Abstract
A carboxypeptidase was isolated from malted barley by affinity chromatography in a yield of approximately 30%. The specific activity was higher than previously obtained for similar enzymes. The enzyme was a dimer where each of the monomers consisted of two peptide chains linked by disulfide bridges. Each monomer contained a single sulfhydryl group, which was inaccessible top-hydroxymercuribenzoate andEllman's reagent when the enzyme was in its native state. The two peptide chains were separated and the N-terminal sequence of each of them determined. The enzyme contained 6 amino sugar residues and 8% neutral sugar. Isoelectric focusing indicated that the enzyme existed in two forms with pI of 5.65 and 5.73, respectively, but N-terminal sequence determination indicated that they had identical peptide chains. Diisopropyl phosphorofluoridate completely inhibited the enzyme while Hg++ only inhibited the enzyme after deprotonation of an ionizable group on the enzyme with a pKa of approximately 6.7.
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Abbreviations
- BS:
-
benzyl succinic acid
- Bz:
-
benzoyl
- Caps:
-
cyclohexylaminopropane sulfonic acid
- CABS-Sepharose:
-
[N-(ε-aminocaproy)-p-aminobenzyl] succinyl-Sepharose 4B
- EDTA:
-
ethylenediamine tetraacetic acid, sodium salt
- DFP:
-
diisopropyl phosphorofluoridate
- FA:
-
furylacryloyl
- Hepes:
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- HPLC:
-
high performance liquid chromatography
- Mes:
-
2-(N-morpholino) ethane sulfonic acid
- p-HMB:
-
parahydroxymercuribenzoate
- PTH:
-
phenylthiohydantoin
- Tris:
-
tris(hydroxy methyl)aminomethane
- Z:
-
carbobenzoxy
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Breddam, K., Sørensen, S.B. Isolation of a carboxypeptidase from malted barley by affinity chromatography. Carlsberg Res. Commun. 48, 217–230 (1983). https://doi.org/10.1007/BF02907768
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DOI: https://doi.org/10.1007/BF02907768