Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2005
Publication Date: 8/1/2005
Citation: Litterer, L.A., Plaisance, K.L., Gronwald, J.W., Somers, D.A. 2005. Assaying uridine diphosphoglucose dehydrogenase in the presence of alcohol dehydrogenase. Analytical Biochemistry. 343(1):192-194. Interpretive Summary: A large proportion of plant cell walls consist of matrix sugars (pectin and hemicellulose). In forages, such as alfalfa, the amount and structure of these compounds plays an important role in determining digestibility by dairy cows. Cell wall matrix sugars also influence the efficiency of converting cell wall biomass into bioethanol. A key protein regulating the synthesis of matrix sugars in cell walls is UDP-glucose dehydrogenase (UGD). Because of its importance, there has been considerable research to measure the level of UGD in plants. We discovered that the assay frequently used to measure this protein is inaccurate because of a contaminant associated with one of the compounds used in the assay. We showed that UGD could be accurately measured if another compound (pyrazole) was added to the assay medium. This research identified a previously unrecognized problem when measuring UGD in plants and describes a simple modification of the assay that results in an accurate measurement. This knowledge will advance research to improve the digestibility of forages by dairy cows and increase the efficiency of bioethanol production from cell wall biomass.
Technical Abstract: UDP-Glc dehydrogenase (UGD, EC 126.96.36.199) activity in crude, desalted plant extracts is frequently assayed using a NAD+-linked spectrophotometric assay. We found that measuring UGD activity in desalted plant extracts using this assay is confounded by the presence of residual ethanol in commercially-available UGD-Glc and the presence of alcohol dehydrogenase (ADH, EC 188.8.131.52) in the extracts. UGD can be accurately measured in desalted extracts if the assay is conducted in the presence of 1 mM pyrazole, an inhibitor of ADH or if contaminating ethanol is removed from UDP-Glc. The validity of UGD assays in the presence of pryazole was confirmed by: (1) inhibition of activity by UDP-Xyl, a feedback regulator of UGD, and (2) quantification of the reaction products by FPLC.