A continuous method for enzymatic assay of sucrose synthase in the synthetic direction

An appropriate method was developed for the continuous assay of sucrose synthase (SS) (EC 2.4.1.13) by spectrophotometry. The uridine 5'-diphosphate derived from sucrose synthesis was stoichiometrically coupled to oxidation of beta-nicotinamide adenine dinucleotide by the enzymes nucleoside-5'-diphosphate kinase (NDPK), pyruvate kinase, and lactate dehydrogenase. Utilization of crude extracts led to a complete masking of SS assay by adenylate kinase, adenosine 5'-triphosphatase (ATPase), and phosphoenolpyruvate phosphatase found in the crude extracts. These interfering enzymes were mostly removed from the crude extracts by using a combination of gel filtration, centrifugation through a selectively permeable membrane (Biomax-100 Ultrafree centrifugal device), and inhibition by the addition of K(2)HPO(4) to the assay buffer. Sensitivity of the SS assay was significantly increased by the inclusion of NDPK and ATP, which are essential to the reaction in the coupling system.