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.