Isolation of the mRNA encoding rat liver catechol-O-methyltransferase

A highly specific, well characterized rabbit antiserum to purified rat liver catechol-O-methyltransferase (COMT; EC 2.1.1.6) and the procedure of polysome immunoadsorption have been used to isolate a messenger RNA which encodes a single polypeptide when translated in vitro. Western blotting and immune fixation have shown multiple active forms of the enzyme to exist; a major, soluble one with MW of 23,000 and pI of 5.2 and another, membrane-bound one with MW of 26,000 and a pI of 6.2 (1). When translated in vitro, the purified message synthesizes a protein of molecular weight (MW) 23,000 and pI 5.2, values in agreement with those for purified enzyme reported by other investigators (2,3). Only the soluble form is seen after in vitro translation; the other immunoreactive proteins possibly arise due to post-translational modifications which do not occur in the lysate; or perhaps another mRNA exists. Cloning of the COMT cDNA will resolve this issue and should be feasible in light of our data indicating that the mRNA isolated here represents 0.46% of total rat liver polyadenylated message.