A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes

Journal Article · Wed May 01 00:00:00 EDT 1991 · Analytical Biochemistry; (United States)

DOI:https://doi.org/10.1016/0003-2697(91)90242-L· OSTI ID:5266783

Salvucci, M E; Crafts-Brandner, S J ^[1]

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1. University of Kentucky, Lexington (USA)

A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, (14C)sucrose phosphate, from unreacted uridine 5{prime}-diphosphate-(14C)glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here the authors show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts.

• OSTI ID: 5266783
• Journal Information: Analytical Biochemistry; (United States), Vol. 194:2; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
TRANSFERASES
RADIOASSAY
ALKALINE PHOSPHATASE
BORONIC ACIDS
CARBON 14 COMPOUNDS
ENZYME ACTIVITY
GLUCOSE
LIQUID COLUMN CHROMATOGRAPHY
PLANTS
TRACER TECHNIQUES
ALDEHYDES
BORON COMPOUNDS
CARBOHYDRATES
CARBON COMPOUNDS
CHROMATOGRAPHY
ENZYMES
ESTERASES
HEXOSES
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHATASES
SACCHARIDES
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques