Functional cysteinyl residues in human placental aldose reductase

Siqi Liu, Aruni Bhatnagar, Ballabh Das, Satish Srivastava

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 m urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.

Original languageEnglish (US)
Pages (from-to)112-121
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume275
Issue number1
DOIs
StatePublished - Nov 15 1989

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Dithionitrobenzoic Acid
Aldehyde Reductase
Ethylmaleimide
Enzymes
NADP
Glyceraldehyde
Catalyst activity
Nicotinamide Mononucleotide
Nitrobenzoates
Sulfhydryl Reagents
Ribose
Mercaptoethanol
Dithiothreitol
Labeling
Urea
Statistical methods
Adenosine Triphosphate
Glucose
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Functional cysteinyl residues in human placental aldose reductase. / Liu, Siqi; Bhatnagar, Aruni; Das, Ballabh; Srivastava, Satish.

In: Archives of Biochemistry and Biophysics, Vol. 275, No. 1, 15.11.1989, p. 112-121.

Research output: Contribution to journalArticle

Liu, Siqi ; Bhatnagar, Aruni ; Das, Ballabh ; Srivastava, Satish. / Functional cysteinyl residues in human placental aldose reductase. In: Archives of Biochemistry and Biophysics. 1989 ; Vol. 275, No. 1. pp. 112-121.
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