Purification of the mannitol-i-phosphate dehydrogenase of Escherichia coli

Abstract

• 1.

  1. Mannitol-i-phosphate dehydrogenase (d-mannitol-i-phosphate: NAD⁺ oxidoreductase, EC 1.1.1.17) was purified by a simple procedure from mannitol-grown Escherichia coli.

• 2.

  2. The active protein was apparently homogeneous in the ultracentrifuge, with a molecular weight of 25 000. At pH 9.0 the $\text{K}{}_{\mathrm{m}}$ for mannitol i-phosphate was $\text{3.1·10}{}^{\mathrm{-4}}\text{M}$, and the $\text{K}{}_{\mathrm{m}}$ for NAD⁺ was $\text{1.4·10}{}^{\mathrm{-4}}\text{M}$. At pH 7.0 the $\text{K}{}_{\mathrm{m}}$ for fructose 6-phosphate was $\text{1.6·10}{}^{\mathrm{-4}}\text{M}$, while that for NADH was $\text{4.2·10}{}^{\mathrm{-6}}\text{M}$. Adenosine phosphates inhibited the enzyme reaction in both directions.

• 3.

  3. In mannitol-grown E. coli, mannitol-i-phosphate dehydrogenase may account for nearly 2% of the total soluble protein.