A specific and sensitive radiometric assay has been developed for the measurement of trehalose&phosphate (trehalose-6-P) synthetase activity. With either [*T]glucose-6-P or UDP-['*C]glucose as substrate, products unique to the synthetase reaction, i.e., trehalose-6-P and trehalose, may be quantitated after charcoal column chromatography by either high-voltage paper electrophoresis or thin-layer chromatography. Some of the characteristics of this assay for synthetase activity which render it more reliable than calorimetric methods based on nucleoside diphosphate production are: (i) absolute product specificity; no other enzyme besides the synthetase catalyzes the synthesis of trehalose-6-P; (ii) greater sensitivity at low levels of product; and (iii) insensitivity to competing side reactions known to interfere with the calorimetric assay (e.g., polyribonucleotide phosphorylase-catalyzed synthesis or polymerization of nucleoside diphosphates).
EXPERIMENTAL PROCEDURES
Chemicals.
[IJ4C]Glucose-6-phosphate (disodium salt), uniformly labeled UDP-[U-14C]glucose, and Aquasol were purchased from New England Nuclear; [U-14C]trehalose was purchased from International Chemical and Nuclear Corporation.
The barium salt of trehalose-6-310