Species Heterogeneity of Pineal Hydroxyindole‐O‐Methyltransferase

Abstract

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.

Synthesis of Melatonin by the Pineal Modified Photoreceptors of Birds Immunocytochemical Localization of Hydroxyindole‐o‐methyltransferase

“…1). A stmtlar molecular weight has been previously reported for chtcken and mammalian HIOMT (8). In addition, a mtnor ~mnunoreacttve pepttde of 28 kDa was observed tn chicken (Fig.…”

Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase

“…This result indicates that excess N-acetylserotonin was not efficiently converted to melatonin by the sheep SNAT plus rice COMT cassette, unlike the Δ83riceSNAT plus rice COMT cassette, suggesting that the COMT-mediated methylation of N-acetylserotonin into melatonin was inhibited by high substrate levels. This type of substrate inhibition has been described in animal ASMTs (Nakane et al 1983). The peak melatonin production of 1465 ng/mL detected after 24 h in the dual sheep SNAT plus rice COMT expression cassette in E. coli was considerably greater than that of other heterologous host plants overexpressing either SNAT or ASMT.…”

“…However, functional expression of animal ASMT in E. coli has been unsuccessful, as the recombinant ASMT proteins are insoluble and inactive (Ben-Abdallah et al 2011). In contrast, the plant ASMT proteins were only soluble and functional as a glutathione S-transferase (GST)-fusion form Zuo et al 2014), but the specific activity of GST-free recombinant ASMT is very low (0.21 pkat/mg protein) relative to that of chicken ASMT (3 nkat/mg protein) (Nakane et al 1983). In contrast to ASMT genes, SNAT genes, the product either from an animal or a plant, were functionally soluble in E. coli, but melatonin production in E. coli by SNAT combined with ASMT has not been possible due to either expression in an insoluble form or inadequate ASMT activity (BenAbdallah et al 2011;Park et al 2013).…”

“…The products of the final two genes in melatonin biosynthesis are serotonin N-acetyltransferase (SNAT) and N-acetylserotonin O-methyltransferase (ASMT), which sequentially catalyze serotonin into melatonin via N-acetylserotonin (Arnao and Hernández-Ruiz 2014). The final gene ASMT has been cloned from a number of animals and plants (Nakane et al 1983;Donohue et al 1993;Kang et al 2011;Zuo et al 2014). However, functional expression of animal ASMT in E. coli has been unsuccessful, as the recombinant ASMT proteins are insoluble and inactive (Ben-Abdallah et al 2011).…”

Generation of the Melatonin Endocrine Message in Mammals: A Review of the Complex Regulation of Melatonin Synthesis by Norepinephrine, Peptides, and Other Pineal Transmitters

“…In the cow, it has been suggested that the subunits could form polymers of a very high molecular weight (Jackson and Lovenberg, 1971). Immunochemical experiments have revealed a large heterogeneity in the protein structure and enzymatic properties among species (Nakane et al, 1983). The protein appears very stable (t 1/2 Ͼ 24 h) (Sugden et al, 1987b;Janavs et al, 1991;Bernard et al, 1993Bernard et al, , 1995Bernard et al, , 1996.…”