Heterogeneity of human erythrocyte transketolase: a preliminary report.
A modified procedure for the preparation of transketolase from erythrocytes by dialysis and column ion-exchange resin chromatography is described. Changes have been made in the resin used, the column dimensions and the elution procedure so as to separate the enzyme with improved resolution, prepare the apoenzyme free of thiamine pyrophosphate and study the kinetics of its activation or reactivation by the coenzyme. On the basis of the elution profile of the enzyme activity from the chromatographic column, two different samples of the transketolase have been isolated, which differ not only in their isoelectric properties, but also in the proportion of the transketolase present in the apoenzyme form. Not only do the apoenzymes isolated from each of the two fractions differ in the way in which they recombine with thiamine pyrophosphate but kinetic analysis of the results shows that each fraction contains at least two variants of transketolase differing in their affinity for thiamine pyrophosphate. Three, probably four, separate variants have been identified which differ in their affinities for thiamine pyrophosphate over a range greater than 10(4). It is concluded that these two fractions of the enzyme must contain different subsets of the eight isoenzymes of transketolase of differing isoelectric points and that some of these isoenzymes must differ also in their affinity for the coenzyme. The implications of these findings for the Blass and Gibson hypothesis about the pathogenesis of the Wernicke-Korsakoff syndrome are considered.
D000425:Alcohol Amnestic Disorder / Q000097:blood / Q000209:etiology; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D003956:Dialysis / Q000379:methods; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014174:Transketolase / Q000302:isolation & purification* / Q000378:metabolism
D001051:Apoenzymes; D007527:Isoenzymes; D014174:Transketolase; D013835:Thiamine Pyrophosphate