Ascorbic acid regeneration by bovine iris-ciliary body
作者:
BodeAnn M.,
GreenEdie,
YavarowChristy R.,
WheeldonSusan L.,
BolkenStephanie,
GomezYvonne,
RoseRichard C.,
期刊:
Current Eye Research
(Taylor Available online 1993)
卷期:
Volume 12,
issue 7
页码: 593-601
ISSN:0271-3683
年代: 1993
DOI:10.3109/02713689309001838
出版商: Taylor&Francis
数据来源: Taylor
摘要:
Ocular tissues appear to require high levels of ascorbic acid and the elucidation of the mechanisms by which those tissues maintain the vitamin in its reduced state remains an important objective. The regeneration of ascorbate from its oxidative by-product, dehydroascorbic acid (DHAA), was studied in bovine iris-ciliary body. Iris-ciliary body was removed by scalpel, weighed, minced, and homogenized in 20 mM MOPS, 62 mM sucrose, and 0.1 mM EDTA at pH 7.0. The homogenate was centrifuged and precipitated with ammonium sulfate such that maximal DHAA reducing activity was enhanced in a 50–75% ammonium sulfate fraction. This fraction was employed for subsequent characterization of DHAA reduction by iris-ciliary body. Results indicate that the iris-ciliary body enzymatically reduces DHAA to ascorbate at a rate significantly greater than can be accounted for by a nonenzymatic glutathione-dependent mechanism. In addition, saturation kinetics are observed, and the enzymatic activity is dependent on protein concentration, DHAA concentration, and reduced glutathione (GSH) concentration. The activity is sensitive to pH, to high temperature, and to digestion by trypsin and is greatest in the presence of both GSH and NADPH. The reducing activity is therefore attributed to one or more proteins that are distinct from the known ascorbate regenerating enzyme, GSH-dependent DHAA reductase (EC 1.8.5.1).
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