Purification and Characterization of Pyrophosphate-

Previously, we reported that inorganic phosphate (Pi) deprivation of Brassica nigra suspension cells or seedlings leads to a progressive increase in the a$-subunit ratio of the inorganic pyrophosphate (PPibdependent phosphofructokinase (PFP) and that this coincides with a marked enhancement in the enzyme’s activity and sensitivity to i ts allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2). To further investigate the effect of Pi nutrition on B. nigra PFP, the enzyme was purified and characterized from Pi-starved B. nigra suspension cell cultures. Polyacrylamide gel electrophoresis, immunoblot, and gel-filtration analyses of the final preparation indicated that this enzyme exists as a heterooctamer of approximately 500 kD and is composed of a 1 :1 ratio of immunologically distinct a (66 kD) and p (60 kD) subunits. l h e enzyme’s a subunit was susceptible to partiai proteolysis during purification, but this was prevented by the presence of chymostatin and leupeptin. In the presence and absence of 5 p~ Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and 7.6, respectively. Maximal activation of the forward and reverse reactions by Fru-2,6-P2 occurred at pH 6.8. l h e potent inhibition of the forward activity by Pi (concentration of inhibitor producing 50% inhibition of enzyme activity [Iso] = 1.3 mM) was attributed to a marked Pi-dependent reduction in Fru-2,6-P2 binding. l h e reverse reaction was substrate-inhibited by Pi (Iso = 13 mM) and product-inhibited by PPi (I5,, = 0.9 mM). l h e kinetic data are consistent with the hypothesis that PFP may function to bypass the ATP-dependent PFP in Pi-starved B. nigra. The importance of the Pi nutritional status to the regulation and predicted physiological function of PFP i s emphasized.