反应详情

The activity of the UdP enzyme was determined in a phosphorolysis test by incubating for 5 minutes at 30° C. the soluble fraction (cell extract) obtained by sonication of a known amount of a suspension of the cell paste and by centrifugation of the homogenate in 100 mM-pH 7 phosphate buffer containing 60 mM of the uridine substrate. The enzyme reaction was blocked by acidification with 0.1N HCl; the suspension was filtered and analysed by RP-HPLC on a C18 column (Hypersyl 100; 4.6×250 mm), eluting under isocratic conditions with a mobile phase constituted by 0.02 M K2HPO4 in methanol-H2O (4:96 v/v) and adjusted to pH 4.5 with NH4OH. The amount of uracil formed in the reaction was determined by reference to a standard curve and the enzyme activity of the cell preparation was calculated in μmol uracil/min/g wet cell paste (units/g). The activity of the PNP enzyme was determined in a phosphorolysis test by incubating for 10 minutes at 30° C. the soluble fraction (cell extract) obtained by sonication of a known amount of a suspension of the cell paste and by centrifugation of the homogenate in 100 mM-pH 7 phosphate buffer containing 50 mM of the inosine substrate. The enzyme reaction was blocked by acidification with 0.1N HCl; the suspension was filtered and analysed by RP-HPLC on a C18 column (Hypersyl 100; 4.6×250 mm), eluting under isocratic conditions with a mobile phase constituted by 0.02 M K2HPO4 in methanol-H2O (4:96 v/v) and adjusted to pH 4.5 with NH4OH. The amount of hypoxanthine formed in the reaction was determined by reference to a standard curve and the enzyme activity of the cell preparation was calculated in μmol hypoxanthine/min/g wet cell paste (units/g).

1. customThe enzyme reaction
2. filtrationthe suspension was filtered
3. washeluting under isocratic conditions with a mobile phase