Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. A multifunctional protein from porcine liver

Abstract

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase from porcine liver have been co-purified more than 500-fold to apparent homogeneity. The inability of three sequential chromatographic procedures followed by affinity chromatography using NADP+- or ATP-substituted Sepharose to resolve the three activities demonstrates that they are physically associated. Molecular weight estimates of the native protein by gel filtration (Mr = 150,000) and by dodecyl sulfate gel electrophoresis (Mr = 100,000) indicate that the native structure is probably a single subunit. Since only one protein band is seen on dodecyl sulfate gels, it is concluded that the three activities are properties of a single polypeptide chain. The kinetic properties of the three activities are described, the most unusual feature being the susceptibility of the cyclohydrolase to competitive inhibition by NADP+, NAD+, ATP, and folate.