Chemistry and Chemical Biology ETDs

Publication Date

12-19-1975

Abstract

Glyoxalase-I has been purified 3OOO-fold from rat erythrocytes V by a procedure employing chloroform-ethanol fractionation and chromatography on CM-Sephadex, Sephaclex GlOO, and DEAE-Sephadex. Two major chromatographi ca lly di sti'nct glyoxa 1 ase-I activity peaks (designated Form I and Form II)i were resolved when a shallow linear salt gradient was used on a DEAE-Sephadex A5O column. Form I showed a specific activity of 861 I.U./mg protein and Form II showed a specific activity of 783 I.U./mg pro,tein although neither was purified to homogeneity. Form I and Form II were distinguishable by disc polyacrylamide gel electrophoresis but indistinguishable kinetically.

Rat erythrocyte glyoxalase,-1 was shown to be similar to yeast glyoxalase-I in that it utilizes the one-substrate pathway, has broad substrate specificity, and employs a rate-determining intramolecular hydride migration.

Metal-free apoenzyme of rat erythrocyte glyoxalase-I was prepared by dialysis against EDTA in the presence of imidazole and glycerol. The apoenzyme thus prepared was: found to have a residua 1 activity of about 1% when assayed in a meta1l-free buffer solution . Activity was restored to the original level upon the addition of Mg2+ Mg2+ gave highest maximum activity (100%), followed by co2+ (85%), Mn2+ (46%), and 2+ 2+ 2+ 2+ . Ni (40%) . Ca , Cu , and Fe: were al so found to activate the apoenzyme slightly (< 10%). The half-saturation concentrations were found to be 66 μM for Mg 2+ , 7.6 μM for Mn 2+ , 3.5 μM for Co 2+ , and 4.5 μM for Ni 2+ from plots of 100/% of V vs. l/[Me2+]

Initial rate studies for the glutathione adducts of methylglyoxal and phenylglyoxal together with their deuterated counterparts in the rat erythrocyte Mn(II)-, Co(II)-, and Ni(II)-glyoxalase-I reactions were carried out. Examination of values of KM, Vmax, Km,h/Km,d and Vmax,h/Vmax,d for enzymes of various metal ions showed marked changes in substrate specificity and isotope effects with different metal ion activated forms of glyoxalase-I.

Language

English

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

David Lee Vander Jagt

Second Committee Member

Illegible

Third Committee Member

Robert Berner Loftfield

Fourth Committee Member

Fritz Schreyer Allen

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