M. A. A. Namboodiri

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

ZEBRAFISH SEROTONIN N-ACETYLTRANSFERASE-2 : MARKER FOR DEVELOPMENT OF PINEAL PHOTORECEPTORS AND CIRCADIAN CLOCK FUNCTION

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish lar...

Ovine Pineal Indoles: Effects of l-Tryptophan or l-5-Hydroxytryptophan Administration

Abstract: l.‐5‐Hydroxytryptophan (l‐5‐HTP) (20 or 200 mg/kg i.p.) but not l‐tryptophan (500 mg/kg i.p.) loading substantially increases serum melatonin in sheep. In the present study we examined the effects of these compounds on pineal serotonin and six serotonin metabolites. l‐Tryptophan failed to increase 5‐hydroxytryptamine (5‐HT; serotonin) or any of its metabolites despite a fivefold increase in pineal tryptophan. In contrast, l‐5‐HTP loading produced a marked increase in pineal 5‐HT and its metabolites, including N‐acetylserotonin (NAS) and melatonin, indicating that an increased synthesis of melatonin is responsible for the increased serum melatonin concentration after loading with this precursor. No change in pineal indoleamine N‐acetyltransferase (NAT) activity was seen. These results are consistent with the suggestion that, during daytime in the sheep, 5‐HT availability may limit the production of melatonin.

Control of the circadian rhythm in pineal serotonin N-acetyltransferase activity: possible role of protein thiol: disulfide exchange

Abstract The activity of pineal serotonin N -acetyltransferase, which is under neural control, is 30- to 70-fold higher at night than during the day. Exposure to light at night causes a rapid (t12 = 3–4 min) decrease in activity. There is reason to suspect this decrease may result from a protein thiol: disulfide exchange.

Multiple Forms of Arylalkylamine N-Acetyltransferases in the Rat Pineal Gland: Purification of One Molecular Form

Rat pineal serotonin N‐acetyltransferase (EC 2.3.1.87) activity is isolated in two molecular forms (Mr≅ 10,000 95,000) by high performance size exclusion liquid chromatography in the presence of ammonium acetate (0.1 M, pH 6.5). In the presence of sodium citrate (0.1 M, pH 6.5), however, it is eluted as a single peak of intermediate size (Mr= 30,000). A highly enriched preparation of one of the molecular forms has been obtained by a two‐step purification procedure involving disulfide‐exchange anion‐exchange chromatography. The N‐acetyltransferase in 250 pineal glands obtained from isoproterenol‐treated rats can be purified about 80‐fold in 1 day; recovery is about 3%. Polyacrylamide gel electrophoresis of the final preparation indicates that a single major b(Mr≅ 11,000) is present; this appears to be serotonin N‐acetyltransferase.

Rapid and reversible activation of acetyl CoA hydrolase in intact pineal cells by disulfide exchange

Abstract Using intact pinealocytes in suspended cell culture it has been determined that acetyl CoA hydrolase activity can be rapidly increased by treatment with cystamine. Similar results are seen with diacetylcystamine, but not with GSSG, penicillamine disulfide, nor with oxidized DTT. The activation of acetyl CoA hydrolase by cystamine is reversible: after cystamine treatment is terminated, enzyme activity decreases slowly in cell culture. It is also possible to reverse the activation by treating homogenates of cystamine-treated cells with dithiothreitol. These observations are consistent with previous findings indicating that pineal acetyl CoA hydrolase activity can be regulated via protein thiol: disulfide exchange. The observations presented in this report also indicate that conditions within the cell allow this type of reaction to take place, and raise the possibility that disulfide exchange mechanisms may be physiologically involved in the intracellular regulation of the activity of this and perhaps other enzymes.

A Simple and Rapid Method for the Purification of Ovine Pineal Arylalkylamine N‐Acetyltransferase

Abstract: A two‐step chromatographic procedure has been developed for the purification of ovine pineal arylalkylamine N‐acetyltransferase (EC 2.3.1.87), based on the principles of disulfide exchange and anion exchange. The enzyme from 20 ovine pineal glands can be purified about 500‐fold in a day; recovery is about 5%. Polyacrylamide gel electrophoretic analysis of the final preparation shows four major bands; one appears to be arylalkylamine N‐acetyl‐transferase.

Inactivation of pineal N-acetyltransferase by disulfide exchange: a possible role of S-S peptides

The enzymatic N-acetylation of serotonin occurs in many tissues (Buda and Klein, 1977; Weber, 1973, Figure 1), and is generally viewed as a common mechanism of serotonin inactivation. In the pineal gland, however, the N-acetylation of the amine is of special importance because it is one step in the conversion of serotonin to melatonin, the hormone which appears to mediate the inhibitory effect of the gland on reproduction (Reiter, 1980).