Kinetic studies with l-type pyruvate kinase from rats either fed a high carbohydrate, low protein diet or starved

Abstract

Rat liver pyruvate kinase (ATP : pyruvate 2- O-phosphotransferase, EC 2.7.1.40) was purified to homogeneity from animals fed a high carbohydrate, low protein diet prior to killing and from animals starved 72 h prior to killing. The liver pyruvate kinase purified from the fed rats always has a specific activity around 220 International Units per mg of protein, whereas the maximum specific activity observed with enzyme purified from the starved rats was 80 units/mg. On polyacrylamide gel isoelectric focusing carried out in the absence of fructose-1,6-bisphosphate, the protein from fed rats resolved into one major band at pH 5.8–6.0 and into an apparent doublet at pH 5.2–5.3. The majority of the protein from starved rats focused into a broad band at pH 5.8–6.0. When isoelectric focusing was carried out in the presence of 5μM fructose-1,6-bisphosphate, both proteins focused at pH 5.2–5.3. The enzymes isolated from both fed and starved rats differed in three kinetic parameters. Firstly, in the difference in specific activities. Secondly, inthe absence of fructose-1,6-bisphosphate the K 0.5 value for phospho enolpyruvate was 0.96 mM for the enzyme from fed rats and was 1.6 mM for the enzyme from starved rats. In the presence of 1 mM fruxtose-1,6-bisphosphate the K 0.5 was 0.15 mM phospho enolpyruvate for both enzymes. Thirdly, at a phospho enolpyruvate concentration of 0.1 mM the K 0.5 values for fructose-1,6-bisphosphate were 0.05 μM for the enzyme from fed rats and 0.13 μM for the enzyme from starved rats. The enzyme from fed rats and from starved rats yielded the same kinetic values for ADP ( K m = 0.4 mM), for phospho enolpyruvate ( K m = 0.15 mM) in the presence of 1 mM fructose-1,6-bisphosphate, for fructose-1,6-bisphosphate ( K m = 1 μM) in the presence of 10 mM l-alanine, and for l-alanine ( K m = 10 mM), when assayed with 1 μM fructose-1,6-bisphosphate and 0.1 mM phospho enolpyruvate.