Isolation of a highly active fructose diphosphatase from rabbit muscle: Its subunit structure and activation by monovalent cations

Abstract

A simple procedure for the purification of fructose 1,6-diphosphatase from rabbit skeletal muscle yields a stable preparation with a specific activity comparable to that of the enzymes isolated from liver and kidney. The isolated enzyme is homogeneous; it shows a single band on electrophoresis in polyacrylamide gels, and sedimentation velocity analysis shows a single, symmetrical species with an s∘20 w value of 7.52 S. The molecular weight from sedimentation equilibrium measurements was calculated to be 142,000, and the enzyme appears to be a tetramer containing subunits of molecular weight of approximately 35,500. The amino acid composition is distinctly different from that of the rabbit liver or kidney enzymes recently isolated in this laboratory, a major difference being that the muscle enzyme is devoid of tryptophan. The muscle enzyme is extremely sensitive to inhibition by AMP and full activity cannot be assayed in crude muscle extracts unless a system to remove AMP is added. For optimal activity a monovalent cation, such as NH 4 + or K +, must also be present. These ions cannot be replaced by Na +, and Li + is inhibitory. The K m values for Mg 2+ and Mn 2+ are increased by NH 4 + which also induces positive cooperativity in the activation by these divalent cations. The sensitivity toward AMP is increased in the presence of NH4 +. Inhibition by AMP shows positive homotropic cooperativity.