Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.
Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.
Ignacak
J
J
Institute of Medical Biochemistry, N. Copernicus Medical Academy, Jagiellonian University School of Medicine, Kraków, Poland.
Gumińska
M
M
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003545:Cysteine; D003600:Cytosol / Q000201:enzymology; D014644:Genetic Variation; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives*
D007527:Isoenzymes; C017842:cibacron blue-sepharose; D012685:Sepharose; D011770:Pyruvate Kinase; D003545:Cysteine