Bovine Pyruvate Kinases III. HYBRIDS OF THE LIVER AND SKELETAL MUSCLE ISOZYMES

Abstract Bovine pyruvate kinases from skeletal muscle and from liver have been hybridized in vitro, producing an equally spaced, five-membered set of electrophoretically distinct species. This set consists of the tetrameric parental forms, designated L4 and M4 for the liver and muscle isozymes, respectively, plus three hybrids designated L3M, L2M2, and LM3 according to their content of type L and type M subunits. The hybrids were separated and isolated by isoelectric focusing. Neither the native enzymes nor the isolated hybrids showed any tendency to exist in more than one electrophoretic form. However, denaturation and renaturation of each isolated hybrid produced a reassortment of its subunits with the subsequent appearance of other members of the electrophoretic set. The subunit designations assigned to the hybrids, and the fact that they are tetramers, is inferred from their equal spacing between the parental enzymes after electrophoresis. In addition, the tetrameric nature of L2M2 was demonstrated directly by showing that it sediments in a sucrose density gradient at a velocity midway between native L4 and M4. Skeletal muscle pyruvate kinase is known to exhibit hyperbolic kinetics (Hill coefficient, nh = 1) and to be unaffected by fructose diphosphate. On the other hand, liver pyruvate kinase exhibits sigmoidal kinetics with varying phosphoenolpyruvate concentrations (nh = 2.4) and is activated by fructose diphosphate. Because the kinetic properties of these two native forms are so very different, we examined the catalytic behavior of the isolated hybrids with the hope of establishing the contribution to total hybrid activity made by the type L and type M subunits. The LM3 hybrid has hyperbolic kinetics with P-enolpyruvate and is unaffected by fructose diphosphate. L2M2, on the other hand, has hyperbolic kinetics with P-enolpyruvate, but has increased activity in the presence of fructose diphosphate. In contrast to these two species, L3M has sigmoidal kinetics with P-enolpyruvate (nh = 1.5) and hyperbolic kinetics only when fructose diphosphate is added. The kinetic properties of the hybrids were compared to mixtures of L4 and M4 having an equivalent ratio of type L to type M subunits. These comparisons showed that the hybrids do not have the same kinetic characteristics as equivalent mixtures of L4 and M4, indicating that the catalytic properties of a subunit are affected by the presence of nonhomologous subunits in the same molecule. It appears that the presence of three type M subunits in the LM3 hybrid, and two in the L2M2 hybrid, induce the L subunits in those species to exhibit hyperbolic kinetics. Conversely, the presence of three type L subunits in the L3M hybrid induces the M subunit in that species to exhibit sigmoidal kinetics. The data obtained in this study suggest that the properties of pyruvate kinase hybrids result from changes in an equilibrium between active and less active forms in agreement with the concerted model of Monod et al. ((1965) J. Mol. Biol. 12, 88–118), although other models may, of course, be equally applicable. It appears that a high proportion of type M subunits favors the active state and produces hyperbolic kinetics, while a high proportion of type L subunits in a hybrid favors the less active state and produces sigmoidal kinetics.