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Hydroxyindole-O-methyltransferase was purified from bovine and chicken pineal glands to apparent homogeneity and their properties were compared. The purified enzymes from both pineal glands differed in electrophoretic mobility and isoelectric point. Sodium dodecyl sulfate gel electrophoresis revealed that hydroxyindole-O-methyltransferase of both bovine and chicken pineals was a dimer consisting of a subunit of molecular weight 39,000. The two enzymes also differed in substrate specificity. Bovine hydroxyindole-O-methyltransferase showed a high specificity toward N-acetylserotonin, whereas chicken enzyme methylated N-acetylserotonin and, to some extent, serotonin and bufotenine. The methylation of the three substrates was probably catalyzed by the same enzyme of chicken pineal, because the ratio of substrate availability did not change throughout the purification steps. Using the purified enzymes, we prepared antibody to both bovine and chicken hydroxyindole-O-methyltransferase. The antibody to bovine enzyme cross-reacted with both avian and mammalian enzymes, whereas the antibody to chicken hydroxyindole-O-methyltransferase reacted with avian enzymes, but far less with mammalian enzymes, indicating an immunochemical difference between avian and mammalian hydroxyindole-O-methyltransferase. The results suggest that the properties of hydroxyindole-O-methyltransferase have changed during the evolutionary development of the pineal glands.

The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression.

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