Low and high temperature asymmetric forms of pig kidney and rabbit muscle phosphofructokinases and reversible, temperature-dependent transitions.

Previous viscometric studies from this laboratory (Johnson, C. S., Vogtmann, L., and Deal, W. C., Jr. (1976) Biochem. Biophys. Res. Commun.73, 391–395) have shown that at 3.5 ° C, pig kidney phosphofructokinase (PFK) is markedly asymmetric and rabbit muscle PFK is moderately asymmetric. The present viscometric and ultracentrifugal studies show that both enzymes are also asymmetric at near-physiological temperatures, that both exist in high-temperature and low-temperature forms, and that the high-temperature forms of both are less asymmetric and more dissociated than the low-temperature forms. The risults also show that the transitions from low- to high-temperature forms are reversible if the exposure to 35 °C is short enough that no irreversible chemical modification occurs. For pig kidney PFK, intrinsic viscosity values of 34.0, 25.6, and 13.8 ml/g were obtained at 3.5, 20 and 35 °C, respectively, whereas rabbit muscle PFK yielded values of 6.9, 6.2, and 5.2 ml/g at the corresponding temperatures. These data clearly show a decrease in asymmetry with increase in temperature. However, both enzymes are still asymmetric at the higher temperature, inasmuch as most globular macromolecules have intrinsic viscosity values in the range of 3 to 4 ml/g, regardless of molekular weight. Studies from 1 to 45 ° C at a fixed protein concentration (4.8 mg/ml) showed that pig kidney PFK has reduced viscosity values of 51.0 ml/g (low-temperature form) and 20.4 ml/g (high-temperature form) in plateau regions of the viscosity graph at the temperature extremes; the mid-point of the transition between the two forms is at about 22–24 °C. Rabbit muscle PFK at 4.2 mg/ml reproducibly gave corresponding reduced viscosity values of 6.9 and 4.8 ml/g for the low- and high-temperature forms, respectively; the transition mid-point between the two forms is at about 16 °C. The first reported sedimentation velocity studies of rabbit muscle PFK at near-physiological temperature (35 °C) show that with near-physiological protein concentration (1.25 mg/ml), the enzyme is in a much more dissociated form, s_20,w(weight average) = 14. 5 S; s_20,w(peak leading edge) = 17 S, than that previously reported at lower temperatures, s_20,w(fastest peak) = 23–30 S. Similarly, the first sedimentation studies on the pig kidney enzyme indicate a lower sedimentation coefficient at 35 ° C (s^0.39%_20,w = 48 S) than at 3.5 ° C(65 S).