The Israeli Journal of Aquaculture - Bamidgeh 55(4), 2003
The 7th Annual Dan Popper Symposium


CHARACTERIZATION OF TWO MELATONIN GENERATING
SYSTEMS IN SEA BREAM:THE PINEAL GLAND
AND RETINAL AANATS


Bina Zilberman-Peled¹, Benny Ron² and Yoav Gothilf¹*

1 Department of Zoology, George S.Wise Faculty of Sciences, Tel Aviv University, Tel Aviv

2 Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, P.O.Box 1212, Eilat 88112, Israel


Abstract

Melatonin is an important component of the vertebrate circadian system.It is produced during the night at two main sites: the pineal gland and the retina. Pineal-derived melatonin is the source for circulating melatonin which provides a hormonal signal that regulates circadian and seasonal physiological rhythms, especially those involving photoperiodicity. Retinal melatonin, on the other hand, is thought to have a local paracrine function related to photic adaptation. The rate of melatonin production in the retina and the pineal gland is controlled by the enzymatic activity of serotonin-N-acetyltransferase (AANAT).

The existence of two AANAT genes, AANAT1 and AANAT2, has been demonstrated in teleost fish. However, the biological significance of this duality is unknown. Toward answering this question, we compared and characterized the two AANATs in the gilthead sea bream (Sparus aurata). cDNA encoding the two sea bream AANATs –sbAANAT1 and sbAANAT2 – has been cloned using PCR amplification. Comparison of their deduced amino acid sequences demonstrate that they share 66% homology. Northern blot analysis demonstrated that AANAT1 is expressed only in the retina, while AANAT2 is expressed only in the pineal gland.

To characterize the enzyme kinetics of the two AANATs, we prepared two recombinant proteins by isolating the full length of the AANAT1 and AANAT2 cDNAs and cloned their open reading frames into a prokaryotic expression vector. The two recombinant proteins were purified and their activity were characterized using a colorimetric assay.

The enzymes demonstrated different activity characteristics, including optimal pH, molarity and temperature. Substrate specificity was determined by incubating the sbAANATs with a variety of substrates. We found that while sbAANAT2 preferentially acetylates indoleethylamines (especially serotonin, the precursor of melatonin), AANAT1 acetylates a variety of arylalay-lamines. Given the differences in the temporal expression pattern and the substrate preferences of the two AANATs, we hypothesize that a gene duplication event results in the formation of two somewhat different systems: AANAT2, expressed in the pineal gland,i s exclusively involved in melatonin production whereas AANAT1, expressed in the retina, may be involved in other biological processes.

*email: YoavG@tauex.tau.ac.il

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