Two methods for determination of transketolase activity

Abstract

Two new optical methods for transketolase activity assay using only one substrate, xylulose 5-phosphate or glycol aldehyde, have been developed. For transketolase activity assay in the first method, it is necessary to add auxiliary enzyme, glyceraldehyde phosphate dehydrogenase. It is not needed in the second method. The range of transketolase concentration in the activity assay is 0.036-0.144 U/ml for the first method and 1.8-6.8 U/ml for the second one.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP‐ribose by the Mn2+‐dependent ADP‐ribose/CDP‐alcohol pyrophosphatase

“…d ‐ribose‐5‐phosphate was measured by kinetic assay of its reaction with 35 μM d ‐xylulose‐5‐phosphate catalyzed by transketolase in 0.6‐ml mixtures containing 25 mM glycylglycine, pH 7.3, 5 mM MgCl 2 , 0.22 mM thiamine pyrophosphate, 5 mM sodium arsenate, 1 mM NAD, 0.1 mg/ml bovine serum albumin, 5 μg/ml transketolase (dissolved in 250 mM glycylglycine, pH 7.3) and 60 m‐unit d ‐glyceraldehyde‐3‐phosphate dehydrogenase (pre‐assayed under these conditions with 80 μM d ‐glyceraldehyde‐3‐phosphate as substrate). Transketolase forms one mole of d ‐glyceraldehyde‐3‐phosphate per mole of d ‐xylulose‐5‐phosphate either by monosubstrate reaction or by bisubstrate reaction with d ‐ribose‐5‐phosphate [22,23]. The formation of d ‐glyceraldehyde‐3‐phosphate is coupled to its arsenate and NAD‐dependent oxidation catalyzed irreversibly by d ‐glyceraldehyde‐3‐phosphate dehydrogenase.…”

“…The increase of A 265 caused by the enzyme itself was determined and used to calculate the true decrease due to AMP deamination. [22,23]. The formation of D-glyceraldehyde-3-phosphate is coupled to its arsenate and NAD-dependent oxidation catalyzed irreversibly by D-glyceraldehyde-3-phosphate dehydrogenase.…”

A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

“…Several assay formats have been developed to probe the substrate tolerance of TK by direct measurement of remaining substrate or formed product. Measurement of chiral product formation by optical activity [25] or ellipsometry [26] is highly dependent on substrate structure and thus not of generic utility. Determination of HPA depletion by near-UV spectroscopic monitoring, [27] enzymatic assay, [28] or HPLC analysis [29] is hampered by low sensitivity or low throughput.…”

Droplet millifluidics for kinetic study of transketolase

“…The results are given in Table III. Several authors have contributed to the determination of the substrate specificity of TK enzymes from different sources (E. coli, S. cerevisiae, G. stearothermophilus) using different assay methods towards various phosphorylated and unphosphorylated aldoses or aldehydes. 4,[14][15][16][17][18][19][20] The activity profiles of TKs are very similar, owing to the strong homology of active sites. Our system yields the same results as those obtained with TK gst using other methods.…”

“…Measurement of chiral product formation by an optical method 16,17 is highly dependent on the substrate structure, and so not of generic utility. Determination of HPA depletion by near-UV spectroscopic monitoring 18 or HPLC (High Performance Liquid Chromatography) analysis [14][15][16][17][18][19][20] is hampered by low sensitivity or low throughput. Colorimetric determination of ketose formation with tetrazolium red-based oxidation is restricted to non-hydroxylated aldehyde acceptors such as propanal.…”