Phosphofructokinase from foot muscle of the whelk, Busycotypus canaliculatum: Evidence for covalent modification of the enzyme during anaerobiosis

Abstract

Phosphofructokinase (PFK) was purified from foot muscle of aerobic and anaerobic (24 h of anoxia) whelks, Busycotypus canaliculatum. Fructose-6-P kinetics were sigmoidal at pH 7.0 with affinity constants, S 0.5, of 2.18 ± 0.10 ( n H = 2.5 ± 0.1) and 2.48 ± 0.13 m m ( n H = 2.7 ± 0.1) for the enzyme from aerobic versus anaerobic muscle. Affinity for ATP, like that for fructose-6-P, did not differ for the two enzymes (0.031 ± 0.003 for the aerobic vs 0.041 ± 0.007 m m for the anaerobic enzyme), but S 0.5 for Mg 2+ was significantly different for the two enzymes (0.060 ± 0.006 vs 0.130 ± 0.020 m m). Whelk muscle PFK was activated by NH 4 +, P i, AMP, ADP, and fructose-2,6-P 2. NH 4 + and fructose-2,6-P 2 were less effective activators of PFK from anoxic muscle, with apparent K a 's 1.6- and 3.5-fold higher for the anaerobic vs aerobic enzyme. Activators decreased S 0.5 for fructose-6-P and reduced n H. With the exception of fructose-2,6-P 2, the effects of activators on S 0.5 were the same for the enzyme from aerobic and anaerobic muscle; fructose-2,6-P 2 at 2.5 μ m reduced S 0.5 by only 3.3-fold for the anaerobic enzyme compared to 5.5-fold for the aerobic enzyme. ATP was a strong substrate inhibitor of PFK; the enzyme from anaerobic muscle showed greater ATP inhibition, with I 50's 1.5- to 2.0-fold lower than those for the aerobic enzyme. The kinetic differences between PFK from anaerobic versus aerobic foot muscle (stronger ATP inhibition and decreased sensitivity to activators for the anaerobic enzyme) were consistent with kinetic differences reported for the phosphorylated versus dephosphorylated forms, respectively, of PFK in other systems. Treatment of PFK from anaerobic muscle with alkaline phosphatase resulted in a decrease in the K a for fructose-2,6-P 2 to a level similar to that of the aerobic enzyme. The physiological stress of anoxia may, therefore, induce a covalent modification of PFK.