Purification and properties of sheep liver phosphofructokinase.
1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4-7mumoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18.5mumoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.
D000227:Adenine Nucleotides; D000255:Adenosine Triphosphate; D000818:Animals; D002021:Buffers; D002845:Chromatography; D002850:Chromatography, Gel; D002951:Citrates; D005632:Fructose; D006600:Hexosephosphates; D008099:Liver / Q000201:enzymology*; D008274:Magnesium; D009243:NAD; D009249:NADP; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds; D012756:Sheep
D000227:Adenine Nucleotides; D002021:Buffers; D002951:Citrates; D006600:Hexosephosphates; D000644:Quaternary Ammonium Compounds; D009243:NAD; D005632:Fructose; D009249:NADP; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium