Separation of two forms of chick 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 24-hydroxylase

We previously reported the development of monoclonal antibodies against chick kidney mitochondrial 1,25-dihydroxyvitamin D3 24-hydroxylase. However, these monoclonal antibodies were able to immunoprecipitate only approximately 25% of total 24-hydroxylase activity present in solubilized preparations, suggesting the existence of different forms of the enzyme. Chromatography of the solubilized preparations on a mono-Q column resolved the 24-hydroxylase activity into two peaks. The protein found in the first peak of enzyme activity (peak 1) did not immunoblot with the monoclonal antibody (IVC2F10) that was raised against the partially purified 24-hydroxylase. However, the protein in the second peak (peak 2) reacted on immunoblot with the same antibody. The enzyme for both peaks had similar kinetics, required adrenodoxin and adrenodoxin reductase, and acted on both 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. It is not yet clear whether these are two distinct enzymes or whether it is the same enzyme complexed with different proteins.