THE PURIFICATION AND PROPERTIES OF BOVINE PINEALS‐ADENOSYLMETHIONINE:N‐ACETYLSEROTONINO‐METHYL TRANSFERASE

Abstract

Abstract— Bovine pineal gland S‐adenosylmethionine: N‐acetylserotonin O‐methyltransferase has been purified about 2800‐fold using cell fractionation, ammonium sulphate treatment, Sephadex G‐200 gel filtration and anion exchange chromatography. The enzyme has been found to be a polymer; the smallest unit observed had a mol. wt. of 21,800 and the other polymers' molecular weights were multiples of this figure. In the gland extract polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. were more abundant than the others; they showed also higher specific activity. One of the products of the reaction, S‐adenosylhomocysteine was found to be a potent inhibitor, whereas the other product, melatonin, did not inhibit the bovine pineal gland enzyme, even at much higher concentrations. Homocysteic acid, cysteic acid, GSG and GSSG inhibited the enzyme. The required concentrations for this effect was 100 times higher than that of S‐adenosylhomocysteine. The addition of GSH to the medium during purification led to complete loss of activity. Adenosine, homocysteine and other thio compounds had little or no effect. The enzyme was found to be activated by its substrates and also by certain anions. Among various organic acid salts, citric acid cycle intermediates were found to be good activators; their nonsubstituted analogues were not as effective. The activator effect of oxaloacetate and bicarbonate was the highest, and was brought about by relatively low concentrations of these anions (1–5 × 10−3 M), hence their effect was considered specific. The degree of activation caused by oxaloacetate was decreased by increasing substrate concentrations and vice versa. The S‐adenosylhomocysteine inhibition could not be reduced by increasing the substrate concentration; S‐adenosylhomocysteine also inhibited the oxaloacetate‐activated enzyme. These observations have been explained by the allosteric behaviour of the enzyme. The kinetic behaviour of various polymers was also investigated. The highest substrate and oxaloacetate activation and the highest S‐adenosylhomocysteine inhibition was observed for polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. The Km values for S‐adenosylmethionine and N‐acetylserotonin calculated for the oxaloacetate activated enzyme were also lower for these polymers than others.

Species Heterogeneity of Pineal Hydroxyindole‐O‐Methyltransferase

“…The specific enzyme activities of the final preparations of bovine and chicken enzymes were 47.1 and 185 nmol melatonin formed/min/mg protein, respectively. The specific activity of our bovine enzyme was comparable with or higher than those reported by other laboratories (Jackson and Lovenberg, 1971 ;Karahasanoglu and Ozand, 1972; Kuwano and Takahashi, 1978). Chicken enzyme had a specific activity four times higher than bovine enzyme.…”

“…Most of these studies, however, were carried out using a crude supernatant of pineal glands. HIOMT has been purified to a high degree only from bovine pineal gland (Jackson and Lovenberg, 1971; Karahasanoglu and Ozand, 1972;Ku-wan0 and Takahashi, 1978).…”

“…There are reports indicating that the properties of HIOMT differ among various animal species (Axelrod and Lauber, 1968;Alexander et al, 1970;Axelrod and Vesell, 1970;Cardinali and Wurtman, 1972). Most of these studies, however, were performed using crude enzyme preparations, a n d HIOMT has been purified only from bovine pineal glands to homogeneity (Jackson and Lovenburg, 1971;Karahasanoglu and Ozand, 1972; Kuwano and Takahashi, 1978). To study the detailed molecular differences of HIOMT among various species, it is essential to obtain a highly purified enzyme from other species of animals.…”

Methoxyindole production by the pineal gland appears to be dependent on the concentration of hydroxy precursors and their affinity for hydroxyindole-O-methyltransferase

“…A number of reports have indicated that several forms of HIOMT exist in the pineal gland (Jackson & Lovenberg, 1971;Karahasanoglu & Ozand, 1972;Cremer-Bartels, 1979) and these various forms may have differing substrate specificities (Cremer-Bartels, 1979). As it has been shown that methoxyindoles other than melatonin exert physiological activity (Mullen, Leone, Hooper et al 1979;Pevet, 1983) and that seasonal variations in synthesis of methoxyin¬ doles exist (Balemans, Bary, van Benthem & Leger¬ stee, 1981) it may be suggested that the pineal gland controls certain physiological functions by variation in methoxyindole synthesis.…”