1,4-alpha-Gluean Phosphorylase from Klebsiella pneumoniae Purification, Subunit Structure and Amino Acid Composition

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Abstract
A 1,4-.alpha.-glucan phosphorylase (EC 2.4.1.1) from K. pneumoniae was purified about 80-fold with an over-all yield greater than 35%. The purified enzyme was shown to be homogeneous by gel electrophoresis at different pH-values, isoelectric focusing, dodecylsulfate electrophoresis and ultracentrifugation. The MW of the native enzyme was determined to be 180,000 by ultracentrifugation studies, in good agreement with the value of 189,000 estimated by gel permeation chromatography. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The MW of these polypeptide chains was found to be 88,000 by dodecylsulfate polyacrylamide gel electrophoresis and 90,000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of 2 polypeptide chains. The enzyme contains pyridoxalphosphate with a stoichiometry of 2 mol/180,000 g protein, confirming that the 1,4-.alpha.-glucan phosphorylase from K. pneumoniae is a dimeric enzyme. The amino acid composition of the enzyme was determined and its correspondence to that of 1,4-.alpha.-glucan phosphorylases from other sources is discussed. The pI [isoelectric point] of the enzyme was 5.3 and its pH-optimum about 5.9. The enzyme is stable in the range from pH 5.9-10.5.