Human Phosphoglycerate Kinase

Human phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) was purified from red blood cells. The stepwise procedures are elimination of hemoglobin from the hemolysate by treatment with ethanol-chloroform, precipitation of the enzyme by ethanol, treatment with carboxymethyl-Sephadex, gel filtration on Sephadex G-75, treatment with diethylaminoethyl-Sephadex, and column chromatography with carboxymethyl-Sephadex. A crystalline preparation was obtained with an over-all yield of about 60%. The sedimentation patterns on analytical ultracentrifugation and the interference pattern of sedimentation equilibrium indicated a homogeneous preparation. The sedimentation constant (s20,w) was 3.35 S at a protein concentration of 0.6%. The molecular weight was estimated as 49,600 by the sedimentation equilibrium method. The enzyme could not be dissociated into smaller subunits by treatment with 5 m guanidine hydrochloride containing 2-mercaptoethanol, with maleic anhydride, or with sodium dodecylsulfate. The enzyme has N-acetylserine as NH2terminal and isoleucine as COOH-terminal. The specific activity of the crystalline preparation was 650 to 700 units per mg at pH 7.5 and at 25°. The enzyme required Mg2+ or Mn2+ for activity.