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Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells.

Abstract
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.
AuthorsKasturi Mukherjee, Swapna Ghosh, Manju Ray, Subhankar Ray
JournalIndian journal of biochemistry & biophysics (Indian J Biochem Biophys) Vol. 39 Issue 5 Pg. 332-41 (Oct 2002) ISSN: 0301-1208 [Print] India
PMID22905379 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Manganese
  • Mercaptoethanol
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Edetic Acid
  • Phosphoglycerate Kinase
  • Magnesium
Topics
  • Adenosine Diphosphate (chemistry)
  • Adenosine Triphosphate (metabolism)
  • Animals
  • Carcinoma, Ehrlich Tumor (metabolism)
  • Chromatography, Gel
  • Chromatography, Ion Exchange (methods)
  • Edetic Acid (chemistry)
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Neoplastic
  • Hydrogen-Ion Concentration
  • Kinetics
  • Magnesium (chemistry)
  • Manganese (chemistry)
  • Mercaptoethanol (pharmacology)
  • Mice
  • Neoplasm Transplantation
  • Nucleic Acid Hybridization
  • Phosphoglycerate Kinase (chemistry, isolation & purification)

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