Isolation and properties of human transketolase

Abstract

Recombinant human (His)(6)-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).

Influence of Transketolase-Catalyzed Reactions on the Formation of Glycolaldehyde and Glyoxal Specific Posttranslational Modifications under Physiological Conditions

“…To our knowledge, the use of glycolaldehyde as an acceptor substrate for human TK has only been described by Meshalkina et al but without tangible substrate characteristics . Though no apparent difference of glycolaldehyde was observed in the presence and absence of TK, the nature of erythrulose formation by the reaction of DHEThDP with glycolaldehyde was unequivocally proven by comparison of the fragmentation spectra of erythrulose formed during model incubations to the reference standard.…”

“…Approximately 0.5 mg of recombinant human transketolase (TK) per g of wet cells was isolated from E. coli with a specific activity of 0.6 U/mg. Properties and enzymatic characteristics were studied previously. ,, To investigate the influence of human TK on the formation of glycolaldehyde and glyoxal specific advanced glycation endproducts (AGEs), model incubations with fructose-6-phosphate (F6P) and ribose-5-phosphate (R5P) in the presence of human serum albumin (HSA) with and without addition of glycolaldehyde were carried out under physiological conditions (pH 7.4, 37 °C) and compared to incubations with inactivated transketolase (iTK). The ratio of the initial reaction educts F6P, R5P, and glycolaldehyde was adapted to the concentrations in vivo , based on our findings (Table ) and the data presented in the literature .…”

“…Fractions containing human TK were pooled, rebuffered to 50 mM triethanolamine and 1 mM dithiothreitol (pH = 7.4), and concentrated by ultrafiltration (Vivaspin MWCO 30000, 4000 rpm, 4 °C). Purity was checked using SDS–PAGE, and protein concentration was determined spectrophotometrically as described in the literature …”

“…Purity was checked using SDS−PAGE, and protein concentration was determined spectrophotometrically as described in the literature. 22 Transketolase Activity. The oxidation of NADH was monitored in a 96 well plate at 340 nm with a Tecan Infinite M200 plate reader (Tecan, Groedic, AUT) at 37 °C for blood samples and 25 °C for recombinant human TK.…”

Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis

“…In order to generate the apo form of mbTK, the cofactors were removed according to the technique described in [ 9 ]. A saturated solution of ammonium sulfate (pH 3.5) was added to the mbTK holoenzyme solution (0.2 mg/mL) in a 10 mM glycylglycine buffer (pH 7.4) at a 2 : 3 ratio.…”