Publication:
Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

dc.contributor.authorJuntratip Jomriten_US
dc.contributor.authorPijug Summpunnen_US
dc.contributor.authorVithaya Meevootisomen_US
dc.contributor.authorSuthep Wiyakruttaen_US
dc.contributor.otherMahidol Universityen_US
dc.contributor.otherAG-BIOen_US
dc.date.accessioned2018-05-03T08:03:44Z
dc.date.available2018-05-03T08:03:44Z
dc.date.issued2011-02-25en_US
dc.description.abstractA sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in 2H2O with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'- 2 H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The 2 H at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the 2 H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of 2 H. The method was highly sensitive that for the aminotransferase with ca. 50kDa subunit molecular weight, only 2mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay. © 2011 Elsevier Inc.en_US
dc.identifier.citationBiochemical and Biophysical Research Communications. Vol.405, No.4 (2011), 626-631en_US
dc.identifier.doi10.1016/j.bbrc.2011.01.080en_US
dc.identifier.issn10902104en_US
dc.identifier.issn0006291Xen_US
dc.identifier.other2-s2.0-79951960222en_US
dc.identifier.urihttps://repository.li.mahidol.ac.th/handle/20.500.14594/11589
dc.rightsMahidol Universityen_US
dc.rights.holderSCOPUSen_US
dc.source.urihttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=79951960222&origin=inwarden_US
dc.subjectBiochemistry, Genetics and Molecular Biologyen_US
dc.titleSensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzymeen_US
dc.typeArticleen_US
dspace.entity.typePublication
mu.datasource.scopushttps://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=79951960222&origin=inwarden_US

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