Studies on adenosine triphosphate transphosphorylases: XI. Isolation of the crystalline adenosine triphosphate-creatine transphosphorylases from the muscle and brain of man, calf, and rabbit; and a preparation of their enzymatically active hybrids

Abstract Detailed procedures are described for the isolation of the crystalline isoenzymes of adenosine triphosphate-creatine transphosphorylase, in good yield, from the muscle and brain of man, calf, and rabbit. The original isolation procedure of Kuby et al. (1) for the rabbit muscle enzyme has been modified to include a batchwise adaptation of a chromatographic step to be described herein for the other muscle types, to permit the isolation of large amounts of a relatively pure preparation for chemical studies. In addition, another convenient procedure is given for the crystallization of the rabbit muscle enzyme from aqueous (NH 4 ) 2 SO 4 solutions, which might be amenable to X-ray structural analyses. Practicable procedures are presented for the preparation, in excellent yields, of the enzymatically active hybrids of the ATP-creatine transphosphorylase, from their component polypeptide chains of the muscle- and of the brain-type enzymes from both man and calf. Descriptions are given for the disruption and annealing of each of the component polypeptide chains of their respective isolated isoenzymes; and a chromatographic system is provided for the separation of the reannealed isoenzymes (with the hybrid included) as native species. Finally, the stability properties of each of the several (six) isoenzymes from the muscle and brain are briefly outlined, in particular, with respect to pH and temperature, to provide a future basis for their physicochemical characterization and comparison. In the case of the human isoenzymes (muscle- and brain-type), a comparative study has been conducted on the kinetics of H + -induced inactivation. These studies illustrate the greater acid lability of the brain type.