Crystallization of spleen-type (M2-type) pyruvate kinase from Rhodamine sarcoma of rats and its properties.

1. Spleen-type (M2-type) pyruvate kinase was purified and crystallized from Rhodamine sarcoma of rats by a procedure involving affinity chromatography on a P-cellulose column and isoelectric separations in the presence and absence of fructose 1,6-diphosphate (FDP). 2. In sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, the purified enzyme migrated, forming a single band corresponding to a molecular weight of approximately 60,000. 3. In molecular-sieve chromatographies of 0.16-0.79 mg/ml of the enzyme in the absence of FDP on a Sephadex G-200 column, the enzyme could exist as monomer, dimer and tetramer, all of which were enzymically active. It existed as a mixture of monomer and dimer at 0.16 mg/ml and as a mixture of dimer and tetramer at 0.79 mg/ml. In the presence of FDP, all the enzyme existed as tetramer at all the concentrations tested, having a molecular weight of approximately 240,000. 4. It was previously reported that the enzyme possessed two different kinds of FDP-binding sites (1st and 2nd sites). It was found that the rate of binding of FDP to the 1st site was remarkably slow, whereas that to the 2nd site was rapid provided that the 1st site had been bound with FDP. The Km values for FDP in its binding to the 1st and 2nd sites were nearly the same, 3-4 x 10-1 M. 5. The FDP bound to the 2nd site was rapidly dissociable, but the FDP bound to the 1st site hardly so. The amount of FDP bound to the 1st site was approximately 2 mol per mol of tetramer. Perhaps, a maximum of 4 mol of FDP per mol of tetramer could be bound. 6. Of the several salts, with which the enzyme was preincubated in the presence and absence of FDP, 0.3 M LiCl, NaCl, KCl, RbCl, NH,CI, (NH3)2S04, KI, KHCO3, and Tris-HCl increased the Vmaac significantly to nearly the same extent.