101] Phosphoglucomutase from yeast

Abstract

Publisher Summary This chapter describes the assay method, purification, and properties of the enzyme, phosphoglucomutase, isolated from yeast. Phosphoglucomutase catalyzes an apparent intramolecular transfer of phosphate between C-1 and C-6 of glucose. The enzyme activity is measured by determining the rate of glucose-6-P formation using a coupled assay involving glucose-6-P dehydrogenase and nicotinamide adenine dinucleotide phosphate kinase (NADP + ). The coupled assay is convenient and also accurate provided that (a) the concentration of phosphoglucomutase is limiting; (b) only the initial rates are recorded; and (c) the change in the absorbance at 340 nm is less than 1.0 per minute. These conditions equate to the formation of less than 0.15 μ mol of glucose-6-P per minute or less than 8% conversion of added glucose-1-P. The steps involved in the purification of phosphoglucomutase are (1) autolysis, (2) heat treatment, (3) ammonium sulfate fractionation, (4) CM-cellulose column chromatography, (5) diethylaminoethyl (DEAE)-cellulose column chromatography, and (6) Sephadex G-100 column chromatography. The isolated protein is homogeneous as judged by ultracentrifugal analysis, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis.