1. The α and β subforms of aspartate aminotransferase were purified from pig heart. 2. The α subform contained 2mol of pyridoxal 5′-phosphate. The apo-(α subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(α subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The β subform had approximately half the specific enzyme activity of the α subform, and contained an average of one active pyridoxal 5′-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5′-phosphate the protein fluorescence of the apo-(β subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.

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