In:
European Journal of Biochemistry, Wiley, Vol. 225, No. 3 ( 1994-11), p. 1073-1079
Abstract:
D‐Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 °C was estimated to be about 0.45 μmol/ml cell water. D‐Arabinono‐1,4‐lactone oxidase, which catalyses the final step in the biosynthesis of D‐erythroascorbic acid, was purified 639‐fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X‐100 solubilisation, ammonium sulphate precipitation, anion‐exchange, hydrophobic‐interaction, gel‐filtration and dye‐ligand chromatographies. Gel‐filtration chromatography and polyacrylamide‐gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin‐containing enzyme. The flavin was not released by treatment with SDS, CCl 3 CO 2 H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40°C and at pH 6.1. The enzyme was stable in the range pH 7.5–10. An apparent K m , value for D‐arabinono‐1,4‐lactone was 44.1 mM. L‐Galactono‐1,4‐lactone, L‐gulono‐1,4‐lactone and L‐xylono‐1,4‐lactone could also serve as substrates. Competitive inhibition was demonstrated with D‐glucono‐1,5‐lactone, L‐arabinono‐1,4‐lactone, D‐galactono‐1,4‐lactone and D‐gulono‐1,4‐lactone. P‐Chloromercuribenzoate, N ‐ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd 2+ , Hg 2+ , Mn 2+ and Zn 2+ exhibited inhibitory effects on the enzyme.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1994.225.issue-3
DOI:
10.1111/j.1432-1033.1994.1073b.x
Language:
English
Publisher:
Wiley
Publication Date:
1994
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
2172518-4
SSG:
12
