Enzymatic production of alpha-D-galactose 1-phosphate by lactose phosphorolysis.

alpha-D-Galactose 1-phosphate (alphaGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of alphaGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells. After purification of the product from the reaction mixture by anion-exchange chromatography and ethanol precipitation, 9.5 grams of highly pure alphaGal1P were obtained from a 1 l reaction volume.

Galactose-1-phosphatase in rat brain.

A prominent galactose-1-phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl-Sephacel, hydroxylapatite, and Sephacryl S-300 columns. The galactose-1-phosphatase was separated from alkaline phosphatase, and from two forms of glucose-1-phosphatase. The three columns gave a 10-fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose-1-phosphate was the best substrate for the purified enzyme, followed by glucose-1-phosphate, which was hydrolyzed 40% as rapidly as galactose-1-phosphate. Galactose-1-phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose-1-phosphate hydrolysis. Mg2+ was required for activity, and supported half-maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L-homoarginine, and L-phenylalanine stimulated enzyme activity. Galactose-1-phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.1S. These values were used to calculate a molecular weight of 50,200 and a frictional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose-1-phosphate concentrations in patients with galactosemia.

Accumulation of galactonate in liver of suckling rats perfused with galactose.

Livers of 14-day-old suckling rats were perfused for 30 min in the nonrecirculating (once-through) mode with media containing 0-60 mM galactose. Galactonate was detected in livers perfused with as little as 1 mM galactose and increased with higher perfusate galactose to 178 nmoles per gram of liver at 60 mM hexose. Tissue galactonate levels were as high as 60% of galactose-1-phosphate which also accumulated under the same conditions. Galactonate was also found to be present in the effluent perfusate. The perfused suckling rat liver appears to be a physiological model for assessing the role of galactose oxidation to galactonate as an alternate metabolic pathway of galactose metabolism.

Lactose and D-galactose metabolism in Staphylococcus aureus. II. Isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate by a specific D-galactose-6-phosphate isomerase.

The inducible D-galactose-6-phosphate isomerase that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus was partially purified from extracts of D-galactose-grown cells. It was shown to catalyze specifically the reversible isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate, the apparent Km values being 9.6 mM and 1.9 mM, respectively. At equilibrium, the ratio of D-galactose 6-phosphate to D-tagatose 6-phosphate was 9.0. The enzyme was not simulated by mono- or divalent cations and was not inhibited by EDTA, but it was inactivated reversibly by the thiol reagent N-ethylmaleimide. Its molecular weight was estimated to be about 100,000 both by gel filtration and by sedimentation in a sucrose density gradient. Data on stability, pH optimum, and inducibility of the enzyme are also presented. An improved procedure for the chemical synthesis of D-tagatose 6-phosphate is described, and resolution of the anomers of D-tagatose 6-phosphate by gas-liquid chromatography is reported.