Molecular cloning, expression, and characterization of myo-inositol oxygenase from mouse, rat, and human kidney.
myo-Inositol oxygenase (MIOX) is a non-heme iron enzyme, which catalyzes the conversion of myo-inositol to d-glucuronic acid, the first committed step in myo-inositol catabolism. Full-length cDNAs of 858bp each coding for 33kDa protein were cloned from kidney cDNA libraries of mouse, rat, and human. The individual clones were expressed in Escherichia coli and recombinant MIOX proteins were purified to electrophoretic homogeneity. A hydrophobic interaction chromatography step yielded multiple conformers, with mouse and human MIOX showing three peaks and rat enzyme revealing two peaks. Individual MIOX peaks exhibited distinct V(max) and K(m) values. Interestingly, upon storage, the 33kDa protein was degraded to a approximately 30kDa truncated protein in each species, and formed small amounts of dimers of identical subunits. While MIOX is a highly conserved enzyme in all mammalian species, the labile nature and tendency to degrade in solution may be the source of significant differences in size previously reported in the literature. Regardless of the source, our results strongly dispel previous conflicting literature reports on the size of the protein and confirm that MIOX is a 33kDa protein.
Arner
Ryan J
RJ
Department of Veterinary Science, Center for Molecular Toxicology and Carcinogensis, 115 Henning Building, The Pennsylvania State University, University Park, PA 16802, USA.
Prabhu
K Sandeep
KS
Reddy
C Channa
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D050562:Inositol Oxygenase; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D010105:Oxygenases; D050562:Inositol Oxygenase