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

Abstract

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent &nbsp;homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH₄)₂SO_{4 &nbsp;}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 <i style="mso-bidi-font-style:normal">p</i>H optimum

(<i style="mso-bidi-font-style:normal">p</i>H 6.0-7.5) for it s enzymatic activity. The apparent <i>K</i>_{<span style="mso-bidi-font-style: italic">m</span>}<i> </i>values of

&nbsp;3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 m<i style="mso-bidi-font-style: normal">M</i> and 0.1 m<i style="mso-bidi-font-style:normal">M </i>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 m<i style="mso-bidi-font-style:normal">M</i> and 0.5 m<i style="mso-bidi-font-style: normal">M</i> respectively. When ATP and 3-PGA act as substrates, ADP, the &nbsp;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.