Cleavage of lens protein-GSH mixed disulfide by glutathione reductase

Satish K. Srivastava, Ernest Beutler

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Abstract

Mixed disulfide complexing the bovine lens erystallins and GSH was prepared by incubating the soluble proteins with GSSG. GSH could be released from the mixed disulfide in the presence of NADPH or NADH. Mixed disulfide was purified using DEAE-Sephadex A-50 column chromatography and was found to be free of glutathione reductase. In such preparation of mixed disulfide NADPH failed to release GSH. However, addition of glutathione reductase along with NADPH released substantial amounts of GSH from the mixed disulfide. Bovine serum albumins also form mixed disulfide with GSH and the mixed disulfide could be cleaved by glutathione reductase. Alkylation of protein sulfhydryl groups by N-ethylmaleimide prior to the addition of GSSG for the preparation of mixed disulfide, almost completely prevents the formation of mixed disulfide indicating that the formation of mixed disulfide involves protein sulfhydryl groups. Evidence is presented that the cleavage of mixed disulfide is not mediated by GSH with cyclic reduction of GSSG by glutathione reductase, but rather proceeds enzymically through glutathione reductase. Using the borohydride method, small amounts of GSH were found to be bound to normal human lens proteins and a substantial amount of GSH was found to be bound to lens proteins from senile cataracts. Only a very small amount of GSH was bound to bovine lens proteins. Glutathione reductase failed to release a significant amount of GSH bound to lens proteins in human cataractous and normal lens. The significantly greater increase in the protein thiols after the borohydride treatment of lens proteins from normal and cataractous human lens and from bovine lens cannot be accounted for by release of GSH bound to proteins. This indicates significant intramolecular and/or intermolecular disulfide bridges in lens proteins.

Original languageEnglish (US)
Pages (from-to)33-42
Number of pages10
JournalExperimental Eye Research
Volume17
Issue number1
DOIs
StatePublished - Oct 1973

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ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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