Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Polymeric forms and subunits.

The molecular weight of a DPN-linked isocitrate dehydrogenase preparation, exhibiting a single major band on polyacrylamide disc gel electrophoresis, has been examined by molecular sieve chromatography. Comparison of the elution volume (V,) of the enzyme with reference proteins of known molecular weight on columns of Sephadex G-ZOO and Sepharose 6B permitted calculation of a molecular weight in the range 290,000 to 365,000. Inclusion of the positive modifier ADP (0.035 mM to 1.0 mM) in the eluting buffer (0.15 M potassium phosphate-O.1 m&r dithiothreitol at pH 7.2) of columns of Sepharose 6B revealed an aggregate of the enzyme of molecular weight 670,000 f 20,000; a species of apparent molecular weight l,lOO,OOO was observed with 0.8 M ammonium sulfate-5 mu potassium phosphate-O.1 m&r dithiothreitol buffer (pH 7.1) containing 0.05 mu to 1.0 mu ADP. Aggregate formation is dependent on protein concentration. Thus, a monomer-dimer equilibrium could be shown when varying amounts of the enzyme were chromatographed on Sepharose 6B in 0.1 mM ADP-0.15 M potassium phosphate-O.1 m&r dithiothreitol at pH 7.2. The limiting values of molecular weight were 330,000 and 680,000 at low and high protein concentrations, respectively. The dissociation constant of the monomer-dimer equilibrium was found to be K = 0.25 f 0.12 mg per ml. The electrophoretic mobility of the enzyme in polyacrylamide gels containing sodium dodecyl sulfate at pH 7.1 or 8 M urea at pH 2.9 indicated polypeptide chains of average molecular weight 40,800 f 500; a value of 40,900 f 700 was obtained by chromatography on Bio-Gel A-5 M in 6 M guanidine hydrochloride. This suggests that the enzymically active monomer of molecular weight 330,000 is composed of eight subunits.