Studies on structure of human erythrocyte phosphofructokinase.

Human erythrocyte phosphofructokinase has been purified to homogeneity and some of its physicochemical properties have been investigated. The enzyme exists in several polymeric states. At high protein concentrations (3 mglml), the predominant form of the enzyme shows a sedimentation coefficient of 57 S. In highly dilute solution (0.3 to 10 pg/ ml), the enzyme is in 12 S to 18 S forms as shown by an “active enzyme” centrifugation technique. Phosphofructokinase dissociates in 6 M guanidinium/HCl to a subunit whose molecular weight is 80,000 as determined by high speed sedimentation equilibrium. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate, however, reveals two protein bands whose molecular weights are 80,000 and 85,000. The 85,000-dalton subunit is similar to the single subunit which occurs in human muscle phosphofructokinase. The 80,000-dalton subunit is designated as human erythrocyte enzyme subunit. The subunits have been separated by DEAE-cellulose chromatography in the presence of 8 M urea, and occur in the ratio of 1:2.9 (muscle:erythrocyte) based upon a comparison of the relative intensity of the stained protein bands on acrylamide gels. Amino acid composition and a tryptic peptide map of human erythrocyte phosphofructokinase are different from those of human muscle phosphofructokinase. Electron microscopic observations show two dominant sets of images. One has a polyhedronal shape which is probably an aggregate of erythrocyte type of subunits. The other aggregate form, which is similar to those of human and rabbit muscle phosphofructokinases, is suggested to be the polymers of muscle type subunits of human erythrocyte phosphofructokinase.