Trehalose Phosphate Synthesis from Uridine Diphosphate Glucose or Guanosine Diphosphate Glucose

Cell-free extracts of a number of mycobacteria catalyzed the synthesis of trehalose phosphate from either uridine diphosphate-d-glucose or guanosine diphosphate-d-glucose and glucose 6-phosphate. A cell-free extract of Mycobacterium smegmatis was separated into two fractions by chromatography on diethylaminoethyl cellulose. Fraction 1 catalyzed trehalose synthesis from GDP-glucose but was relatively inactive with UDP-glucose. However, when Fraction 2 was added to Fraction 1, UDP-glucose was able to serve as an effective glucosyl donor for trehalose synthesis. Under these conditions, GDP-glucose was still active, but less so than with Fraction 1 alone. Fraction 2 had no detectable enzymatic activity and had the following properties. (a) It was heat-stable and gave a typical RNA ultraviolet absorption spectrum, (b) it was orcinol-positive and diphenylamine-negative, and was inactivated by incubation with RNase, (c) it was retained on a 50,000 molecular weight filter but passed through a 100,000 filter. Fraction 2 could be replaced by crude α-lactalbumin but not by purified lactalbumin, lysozyme, serum albumin, or a number of other proteins. The active component of crude lactalbumin, isolated by phenol extraction, was found to be susceptible to RNase. Although Fraction 2 was quite active in causing UDP-glucose utilization, it could be replaced to a greater or lesser extent by polyuridylic acid, the RNA fraction of α-lactalbumin, or the RNA fraction of M. smegmatis. The activity of each of these was destroyed by treatment with RNase. Purified yeast RNA, yeast transfer RNA, polyadenylic acid, UTP and UMP were all inactive or only slightly active when tested at various concentrations. UDP was somewhat active at high concentrations, apparently because of polynucleotide phosphorylase. The dephosphorylated products formed from either UDP-glucose-14C and Fractions 1 and 2, or GDP-glucose-14C and Fraction 1 were identified as α,α-trehalose since they were stable to alkali, had the same chromatographic mobility as trehalose in several solvents, were hydrolyzed to glucose by a specific trehalase, and their octaacetate derivatives cocrystallized to constant specific activity with authentic α,α-trehalose octaacetate.