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A chromatographic method has been developed which permits the isolation of phosphorylated and dephosphorylated enzymatically active forms of phosphoglucomutase. The enzyme, obtained from rabbit muscle, is chromatographed at pH 7 on carboxymethyl-cellulose, eluting with a linear gradient of sodium phosphate buffer. Inactive protein, if present, passes through the column and is separated from the active enzyme forms which are subsequently eluted.Chromatographed enzyme can be stored at approximately 0°as a slurry in pH 5-buffered ammonium sulfate; or, alternatively, in liquid nitrogen as frozen pellets of neutral buffered solutions. That a homogeneous enzyme of high purity can be obtained by such chromatography was evidenced by the constant specific enzymatic activity exhibited by the fractions belonging to a single peak, by the enzyme's subsequent elution as a single peak upon repeated chromatography, and by its homogeneity in equilibrium ultracentrifugation. The phosphorylated and dephosphorylated nature of the separated phosphoglucomutase components was identified by 32P-substrate labeling experiments. Amino acid analyses were performed, and the composition of the chromatographed phosphoenzyme was compared with that published for phosphoglucomutase in earlier reports.Recent studies concerning the relationship of structural characteristics of phosphoglucomutase to its activity (Koshland etal., 1962), in some cases involving rather small but measurable changes, emphasized the desirability of finding a reproducible method for obtaining enzyme samples of the highest purity. Moreover, according to the mechanism of action proposed by Najjar and Pullman (1954), this muscle enzyme must exist in two catalytically active forms, one a phosphoprotein and the other a nonphosphorylated species. A separation of these two forms would be

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A molecular weight of 183,000 + 5% and a sedi-the enzyme is a hexamer composed of seemingly identical subunits. The amino acid composition was also determined. Results of p-mercuribenzoate titration studies imply that twothirds of the sulfhydryl groups (possibly two of the three cysteinyl residues calculated per subunit) are shielded in the native form and that the enzyme contains no disulfide bridges. However, it appears that a sulfhydryl group is nor required for enzymatic activity.with the determination of the spo,w value at infinite dilution, the molecular weight, subunit structure, and the amino acid composition of this aminotransferase. Information regarding aggregation properties of the enzyme, identification of its N-terminal amino acid residue, and the effects of thiols and sulfhydryl reagents on the enzyme are also included. Experimental SectionEnzyme Purification and Assay. The crystallization procedure and assay for transaminase B were as previously described by Coleman and Armstrong (1971). Enzyme preparations with specific activities of 3500-4000 pmol of isoleucine transaminated per hr per mg of protein were used in this study. Protein was determined by the method of Lowry et al. (1951). S a l m o n e l l a T R A N S A M I N A S E BMaterials. The following thiol compounds and sulfhydryl reagents were used : 0-mercaptoethanol, reduced glutathione, p-mercuribenzoate, and N-ethylmaleimide (Sigma Chemical Co.); and 24odoacetamide (Eastman).Proteins that served as standards in the sodium dodecyl sulfate polyacrylamide electrophoretic or Sepharose 6B chromatographic studies included : horse heart cytochrome c, bovine trypsin, bovine liver catalase, bovine fibrinogen, and rabbit muscle aldolase (Sigma Chemical Co.) ; bovine chymotrypsinogen A, rabbit muscle aldolase, and ovalbumin (Pharmacia Fine Chemicals, Inc.); horse heart myoglobin and bovine serum albumin (Schwarz/Mann) ; and yeast alcohol dehydrogenase (Miles-Seravac, Ltd.). Other compounds and materials used included : sodium dodecyl sulfate (99 % purity) and 2,4-dinitrophenyl (Drip)' amino acid standards (Sigma Chemical Co.) ; guanidinium chloride (99 purity), methylenebisacrylamide and silica gel chromagram sheets (Eastman) ; Temed, ammonium persulfate, acrylamide, Bromophenol Blue, and a-ketoglutarate (Matheson Coleman and Bell); Coomassie Brilliant Blue (Mann Research Laboratories); thioglycolic acid (Fisher Scientific Co.) ; Sepharose 6B (Pharmacia Fine Chemicals, Inc.) ; analytical grade KC1 (Mallinckrodt); and Baker-flex cellulose sheets (J. T, Baker Chemical Co.).

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