Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells.
3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata 700 032, India.
Kasturi
K
Mukherjee
Swapna
S
Ghosh
Manju
M
Ray
Subhankar
S
Ray
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D009368:Neoplasm Transplantation; D009693:Nucleic Acid Hybridization; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000302:isolation & purification*
D008345:Manganese; D008623:Mercaptoethanol; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D010735:Phosphoglycerate Kinase; D008274:Magnesium