Studies on the fatty acid inactivation of phosphofructokinase

Investigation of phosphofructokinase in normal and regenerating livers led to the discovery of an inactivating factor in the extracts of these livers. The inactivating factor was found to be a mixture of free fatty acids. The fatty acid compositions of the normal and regenerating livers are the same, but the concentrations of most of the fatty acids are at least 3 to 4 times higher in the latter. Inactivation of phosphofructokinase by palmitate and oleate was investigated using purified rabbit muscle enzyme. Incubation of the enzyme with palmitate (250 μM) or oleate (50 μM) resulted in rapid inactivation of the enzyme with biphasic curves. The concentrations of oleate and palmitate required to produce 50% inactivation of the enzyme were 35 μM and 75 μM, respectively. Fructose 6 P (0.5 mM), MgATP, (1 mM), fructose 1,6 P₂ (1 mM), AMP (1 mM), and cyclic adenosine 3':5' monophosphate (20 μM) protected the enzyme against inactivation when these metabolites were incubated with the enzyme before the addition of fatty acid. Bovine serum albumin (100 μM) and β cyclodextrin (0.25 mM) also protected the enzyme against the inactivation. However, if the enzyme was inactivated by fatty acid, subsequent addition of the above metabolites or bovine serum albumin did not reactivate the enzyme. Binding studies with [³H]oleate revealed at least three types of binding sites. The first site binds 2 to 4 mol of oleate/mol of enzyme. Oleate binding to this site did not seem to affect the enzyme activity. The second binding site binds 5 to 15 mol of oleate/mol of enzyme resulting in complete loss of the activity. This is followed by an increase in oleate binding to the third site of the enzyme. Sucrose density gradient centrifugation of oleate inactivated enzyme indicated that the enzyme dissociated to the dimeric form. Similarly, centrifugation of [³H]oleate treated enzyme revealed that all polymeric forms of phosphofructokinase bound approximately 6 to 8 mol of oleate/mol of enzyme. In the presence of fructose 6 P, oleate is bound to the polymers to a lesser degree and therefore protects against the fatty acid inactivation. Various polymers which are cross linked with dimethylsuberimidate are also inhibited by oleate.