Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad, Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with P-32-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that P-32 was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.