Hello Kedar,
I assume the question is about an UDP-sugar utilizing glycosyltransferase where UDP will be a product? If so I would certainly try coupling the reaction to the pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities and to measure continuously the reaction by spectrophotometer (at 340 nm). UDP (product of glycosyltransferase rxn) should react with PEP (via PK), and then the resulting pyruvate will be metabolized with NADH to lactate via LDH. Thus, in the assay, one would need to have the glycosyltransferase, the UDP-sugar, the acceptor for UDP-sugar, PEP, NADH and both PK and LDH, Of course the accuracy of the method would be best for purified glucosyltransferase.
I assume here (I do not know for certain) that PK can react with UDP (PK normally reacts with ADP). Nevertheless it's worth a try, imho.
Regards
Leszek
I published a method that will allow you to detect the UDP or GDP product of the reaction with nanomolar sensitivity using fluorescence. The assay requires the enzyme polynucleotide phosphorylase, which I can give you, RiboGreen RNA dye from Life Technologies, and polyA RNA (from e.g. Sigma) for detecting UDP. No RNA is needed for detecting GDP. The principle of the assay is to convert the nucleoside diphosphate into double-stranded RNA.
High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity
Analytical Biochemistry, Volume 415, Issue 2, 15 August 2011, Pages 190-196
We used fluorescent labelled substrate to measure the glycosyltransferase activity by HPLC with fluorescent detector and NH2 column. Fluorescent labelled substrates are commercially available for most of the glycosyltransferases.
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