We have reviewed the allosteric regulatory properties of skeletal muscle phosphofructokinase and recent results on the phosphorylation of this enzyme. The number and affinities of various ligand binding sites are described, and a simple three state model is presented to explain the kinetic and ligand-binding properties of the enzyme. Data describing a lack of fit to a concerted transition model are presented. The widespread occurrence of partial phosphorylation of phosphofructokinase at a specific site near the carboxyl terminus is documented, as well as the lack of significant kinetic consequences of such phosphorylation.

Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered.

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.

The distribution of phosphofructokinase (PFK) in gray and white matter regions of the rat nervous system was evaluated. Determinations of PFK activity revealed that cell body enriched regions (sensorimotor cortex) had a significantly higher level of activity than axonal regions (sciatic nerve, dorsal roots, and optic nerve). The level of PFK activity was also significantly higher in central axons (optic nerve) than in peripheral axons (sciatic nerve). Differences in PFK activity could be largely attributed to differences in tissue content of the enzyme rather than to differences in the types of PFK isozymes present. Cortex contained significantly larger amounts of PFK relative to total protein than did peripheral nerve. However, purification of PFK revealed that all three of the PFK isozymes, C (86 kd), A (84 kd), and B (80 kd), were present in both cortex and sciatic nerve. Both SDS/PAGE and immunoblotting studies using PFK isozyme-specific antibodies demonstrated that the relative proportions of the three PFK isozymes were similar in cell body and axonal regions of the nervous system. The PFK-C and PFK-A isozymes each comprised about half the total and only small amounts of the PFK-B isozyme were present in both regions. However, immunoprecipitation experiments suggested that quantitatively different proportions of the possible PFK hybrids (tetramers) may be distributed between axonal and cell body regions. The transport of PFK was examined in this study and PFK was identified in slow component b (SCb) of axonal transport. SCb moves at a rate of 2-4 mm/day in rat axons and is known to contain several other enzymes of intermediary metabolism as well as actin. The finding that PFK, the rate limiting enzyme in glycolysis, is present in SCb lends support to the hypothesis that glycolytic enzymes are not freely diffusing proteins in axons but, instead, are present as organized assemblies that have long-term, yet flexible, associations with structural elements of the cytoplasm.

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