A detailed quantitative study of the specificity of this enzyme to nucleoside diphosphates serving as glucosyl acceptors has now been made using a highly purified sucrose synthetase from sugar beet root6, which is free of invertase and phosphatase activities. The results presented in Table 1 show that TDP is a very efficient acceptor whereas ADP is much less effective in this case than with the sweet corn enzyme system4. Chromatographic analysis7 of reaction systems containing UDP, TDP and ADP, as described in Table 1, indicated the appearance of new nucleotide components which behaved in the same way as authentic UDP-glucose, TDP-glucose and ADP-glucose, respectively. Table 1. EFFECT OF ISTUCLEOSIDE DIPHOSPHATES ON SUCROSE CLEAVAGE BY SUCROSE SYNTHETASENucleotide Fructose liberated (?moles/m/./h) Relative activity UDP 129 100TDP 067 52 ADP 020 16CDP 015 12 GDP 008 6None 002 ? The reaction mixture contained: tris, 25 mM; sodium acetate, 20 mM; mono-sodium phosphate, 20 mM (pH 60); sodium fluoride, 20 mM; sucrose, 200 mM; EDTA, 15 mM; nucleotide, 25 mM; purified enzyme, 22 ?g protein/ml.; and bovine serum albumin, 50 ?g/ml. Incubation was at 37. The amount of fructose liberated was measured as described previously2.From a reaction mixture similar to that described in Table 1, and containing TDP as the nucleotide, the new sugar nucleotide component was quantitatively isolated by paper chromatography, analysed chemically and enzymatically, and shown to be TDP-D-glucose, using the chemical and enzymatic procedures already described8. A reaction mixture which contained tris buffer, 120 mM at pH. 72; fructose, 60 mM; EDTA, 6 mM; sodium fluoride, 30 mM; TDP-glucose, 6 mM, and purified enzyme, 144 ?g/ml., contained, after incubation for 3 h at 37, about 3 mM disaccharide which was identified as sucrose by paper chromatography, specific colour reactions and enzymatically2. Kinetic investigations2 have clearly shown that UDP and TDP as well as UDP-glucose and TDP-glucose are competing substrates for the same site of the transgluco-sylase. The Vmax value for sucrose synthesis at pH. 72 with UDP-glucose as the substrate was 37 times higher than that observed for TDP-glucose. The Vmax value for sucrose cleavage at pH. 60 using UDP as the glucosyl acceptor was 17 times higher than that observed when TDP was used. The apparent Km values obtained for the uridine and thymidine nucleotides were of the same order of magnitude (Table 2).Table 2. APPARENT Km VALUES FOR THE URIDINE AND THYMIDINE NUCLEOTIDES Substrate pH of assay Km (M)UDP 60 77 x 10?5 TDP 60 94 x 10?5UDP-glucose 72 27 x 10?4 TDP-glucose 72 95 x 10?5Sucrose cleavage was followed by measuring the appearance of free fructose in a system containing 025 M sucrose and varying concentrations of nucleoside diphosphate. Sucrose synthesis was assayed by measuring the appearance of UDP, or of free glucose liberated by addition of yeast invertase, in a reaction mixture containing 10 mM fructose and varying concentrations of sugar nucleotides. Analyses were carried out as described elsewhere2. TDP-glucose is an intermediate in the biosynthesis of TDP-L-rhamnose and TDP-D-galactose, which serve as glycosyl donors in the biosynthesis of complex sacchar-ides9'10. It is thus possible that formation of TDP-glucose by the sucrose synthetase of plant storage tissues, where sucrose is found in high concentration, is a significant pathway for the supply of glycosyls to various biosynthetic reactions occurring in these tissues. Recent experiments in our laboratory have shown the presence of several thymidine diphosphate sugar derivatives which occur naturally among the soluble nucleotides obtained from sugar beet roots.This work was supported by research grant FG-IS-141 from the U.S. Department of Agriculture.