Quantitative characterization of homo- and heteroassociations of muscle phosphofructokinase with aldolase

Dissociation of purified phosphofructokinase accompanied with inactivation was analyzed in the absence and presence of aldolase and the data were comp ared with those obtained with muscle extract. The kinetics of the decrease in enzymatic activity was highly dependent on the dilution factor in both c ases, but the inactivation appeared to be biphasic only with extract. The i nactivation of the phosphofructokinase was impeded by addition of excess of aldolase. Time courses of kinase inactivation were fitted by alternative k inetic models to characterize the multiple equilibria of several homo- and heterooligomers of phosphofructokinase. The combination of modeling data ob tained with purified and extract systems suggests that aldolase binds to an intermediate dimer of phosphofructokinase and within this heterocomplex th e kinase is completely active. The intermediate dimer is stabilized by asso ciation with microtubules and the kinase activity decreased due to dilution can be recovered by addition of excess aldolase. In extract, the phosphofr uctokinase is of sigmoidal character (Hill coefficient of 2.3); the additio n of excess exogenous aldolase to phosphofructokinase resulted in heterocom plex formation displaying Michaelian kinetics. The possible physiological r elevance of heterocomplex formation of phosphofructokinase in muscle extrac t is discussed. (C) 2000 Elsevier Science B.V. All rights reserved.