Multifunctionality of the Enzyme Involved in the 2,3-Diphosphoglycerate Metabolism of Pig Erythrocytes

Abstract

In a continuing study of the enzymes involved in the 2, 3-diphosphoglycerate metabolism of mammalian erythrocytes, we report that 2, 3-diphosphoglycerate in pig erythrocytes is probably metabolized by one multifunctional enzyme. Diphosphoglyceromutase, 2,3-diphosphoglycerate phosphatase, and phosphoglycero-mutase were simultaneously purified from pig erythrocytes. Three fractions (peaks I, II, and III) which had all three activities in different ratios were obtained by column chromatography. Peak I was extremely rich in phosphoglyceromutase, containing more than 90% of the total activity of this enzyme. In contrast, peak III was active in metabolizing 2, 3-diphosphoglycerate, containing about 95% of both the diphosphoglyceromutase and the 2, 3-diphosphoglycerate phosphatase activity. It seems likely that peak I functions as phosphoglyceromutase and that peak III functions in the 2, 3-diphosphoglycerate metabolism. The homogeneity of peak III was established by disc gel electrophoresis in the presence and the absence of sodium dodecyl sulfate, as well as by ultracentrifugation. The three activities of peak III were lost at the same rate on thermal inactivation. These results indicate that the two enzyme activities for metabolizing 2, 3-diphosphoglycerate, which were believed to be due to different proteins, are attributable to one protein, along with some phosphoglyceromutase activity. The diphosphoglyceromutase activity of this protein was inhibited by the product, 2, 3-diphosphoglycerate, as well as by hydroxypyruvate phosphate, 2-phosphogly-colate, inorganic phosphate, and bisulfite. The 2, 3-diphosphoglycerate phosphatase activity was enhanced by 2-phosphoglycolate and hydoxypyruvate phosphate, and by the coexistence of chlorine ion and bisulfite, while it was inhibited by monophosphoglycerates. The native peak III protein had a molecular weight of 59,000 as determined by equilibrium centrifugation. Disc gel electrophoresis in the presence of sodium dodecylsulfate yielded a single protein band with a molecular weight of 28,000, indicating that this protein was composed of two subunits with a similar molecular weight. Occurrence of multifunctional proteins in pig erythrocytes is compared with that in human erythrocytes from the standpoint of the universal existence of such proteins.