myo-Inositol oxygenase from hog kidney. I. Purification and characterization of the oxygenase and of an enzyme complex containing the oxygenase and D-glucuronate reductase.

Homogeneous myo-inositol oxygenase (EC 1.13.99.1) has been obtained in 45% yield by a 550-fold purification from a centrifuged homogenate of hog kidney. Of particular importance in developing the purification procedure was the finding that although the enzyme becomes less catalytically active during purification, it could be reactivated by incubating with 1 m~ Fe(II) and 2 m~ cysteine. The molecular weight of the homogeneous oxygenase, as determined by 3 independent methods, is 65,000 2 1,000, and no evidence could be found for the existence of any smaller subunits. The specific activities of homogeneous preparations vary, and this was found to correlate with the iron content; the most active preparations have 4 atoms of iron/ 65,000 daltons. The homogeneous oxygenase has kinetic characteristics (pH maximum at 6.0) different from those (pH maximum at 8.0) of the oxygenase present in crude extracts. By modifying the purification procedure, another form of the oxygenase, which retains the kinetic characteristics of the enzyme in the homogenate, was purified 300-fold in 36% yield. Analytical gel electrophoresis indicates that 1 migrating species accounts for about 90% of the protein in the purified preparation. This labile species, which has a molecular weight of approximately 250,000, was shown to be an enzyme complex, which contains not only the oxygenase, but also D-glucuronate reductase (EC 1.1.1.19) and at least 2 other unidentified proteins. In its native state, the oxygenase is believed to exist as part of this complex.