Journal of Pineal Research 3:389-395 (1986)

Purification of Rat Pineal Hydroxyindole-0- Methyltransferase Using S-Adenosyl-L-

Homocysteine Agarose Chromatography

David Sugden, Pierre Voisin, and David C. Klein Section o n Neuroendocrinology, Laboratory of Developmental Neurobiology,

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Rat pineal hydroxyindole-0-methyltransferase (HIOMT; EC 2.1.1.4) was purified by affinity chromatography using an S-adenosyl-L-homocysteine agarose column. This single-step procedure, which is rapid, simple, and applicable to small quantities of tissue, gave a large enrichment of a protein (M, - 38,000) identified by SDS-PAGE and silver staining. The amino acid composition of rat HIOMT was generally similar to that of the bovine enzyme, although some differences were apparent. This method will be valuable in isolating sufficient rat HIOMT to enable its primary amino acid sequence to be determined.

Key words: affinity chromatography, proteinlenzyme purification, amino acid analysis, indoleamines

INTRODUCTION

Hydroxyindole-0-methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the final step in the synthesis of the pineal hormone melatonin fAxelrod and Weissbach, 19601. Like arylalkylamine N-acetyltransferase (EC 2.3.1.87), the rate-limiting enzyme in this pathway, HIOMT is neurally regulated via sym- pathetic neurons originating in the superior cervical ganglia [Sudgen and Klein, 19831.

Although HIOMT has been isolated from large quantities of bovine and chicken pineal tissue using multiple steps Uackson and Lovenberg, 1971; Kuwano and Takahashi, 1978; Nakane et al., 19831, a simple purification method is not available for small quantities of tissue. The present report describes a one-step method for the isolation of rat pineal HIOMT using S-

Received March 3, 1986; accepted May 12, 1986.

Address reprint requests to Dr. David C. Klein, Building 10, Room 8D-42C, National Institutes of Health, Bethesda, MD 20892.

0 1986 Alan R. Liss, Inc.

390 Sugden, Voisin, and Klein

adenosyl-L-homocysteine agarose chromatography and reports the amino acid composition of the purified material.

MATERIALS AND METHODS

S-adenosyl-L-homocysteine (AdoHcy) covalently linked to acetamido- hexyl-agarose via a secondary amido linkage was obtained from Bethesda Reserach Laboratories (Gaithersburg, MD). ['4C-Methyl]-S-adenosyl-L-methi- onine (specific activity 50 Ci/mmol) was purchased from ICN (Irvine, CA) and N-acetylserotonin and S-adenosyl-L-methionine, chloride salt (AdoMet), were from Sigma Chemical Company (St. Louis). Chemicals for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad (Richmond, CA).

Pineal glands were rapidly removed from adult male Sprague-Dawley rats (Zivic Miller Laboratories, Allison Park, PA) killed between 1200 and 1600 h, frozen immediately on dry ice, then stored (-30 "C) until used. Pineals (1 gland/lO PI) were sonicated briefly on ice in ammonium acetate (10 mM, pH 6.8) containing dithiothreitol (1 mM). The sonicate was centri- fuged (2 min, 4"C, 12,00Og), and the resulting supernatant was used in the purification procedure. HIOMT activity was assayed as described previously [Sugden et al., 19831. Polyacrylamide gel electrophoresis was performed using a modification of the method of Laemmli [1970] with the aid of a BRL model V16-2 unit. Gel concentrations were 3% acrylamide for the stacking gel and 10% for the resolving gel. The gels were fixed and stained with silver or Coomassie blue. Proteins were electroeluted from unfixed, Coomassie blue-stained gels using an ISCO electroelution apparatus (model 1750, ISCO, Inc., Lincoln, NE) at 1 watt for 18 h [Hunkapiller et al., 19831.

RESULTS

Essentially all (> 95%) of the HIOMT activity loaded was bound to the AdoHcy-agarose column. A considerable proportion of the other soluble pineal proteins (average 50%) were eluted during the initial washing of the column (Fig. 1.) HIOMT bound equally well at pH 6.8 (ammonium acetate, 10 mM) and pH 7.4 (sodium phosphate, 5 mM). Furthermore, it was not necessary to stop the flow of the column to promote maximal binding [Hurst et al., 19831. A high salt wash (NaCl, 1 M in ammonium acetate, 10 mM, pH 6.8) eluted virtually all (> 95%) of the bound protein but only approximately 20% of the bound HIOMT activity (Fig. l), presumably that fraction not specifically bound to AdoHcy. Subsequent washes with AdoMet (5 pM to 1 mM) did not elute detectable amounts of HIOMT activity or protein. Exten- sive dialysis and concentration (> 80-fold) of these fractions revealed only minor amounts of HIOMT activity (c 1 % of the total bound). Approximately 20-30% of the HIOMT activity initially bound on the column was eluted at low pH (KC1 0.1 M/HCI 0.1 M, pH 2.4). Recovery was variable, probably because of the instability of the enzyme under these conditions, even though the eluate was rapidly neutralized with sodium phosphate buffer (0.4 M, pH 7.9). A summary of a typical purification run is given in Table 1. Usually, the

Purification of Rat HIOMT 391

I -t t

0 10 20

ELUTION VOLUME Iml)

Fig. 1. Purification of rat pineal HIOMT. Rat pineal extract from 20 glands was applied to a column (0.35 . X 2.5 cm) of AdoHcy-agarose equilibrated with ammonium acetate 10 mM, dithivthreitol 1 mM, pH 6.8, at a rate of - 10 ml/h. The column was washed sequentially with NaCl (1 M in loading buffer), S-adenosyl-L-methionine (1 mM in loading buffer), loading buffer (ammonium acetate 10 mM, dithiothreitol 1 mM, pH 6 4 , and 0.1 M KCl/O. 1 M HCl, pH 2.4. Fractions eluted by acid (0.43 ml) were collected into tubes containing sodium phosphate buffer (0.4 M, pH 7.9, 70 PI). Protein (0) and HIOMT activity (0) were assayed as described in “Materials and Methods.”

TABLE 1. Affinity Purification of Hydroxyindole-0-Methyltransferase

Volume Total activity Total protein Specific activity Yield Purification step (ml) (nmollh) (ms) (nmol/h/mg) (”/.I Crude supernatant 0.2 3.02 1.81 1.67 100 AdoHcy-agarose 4.3 0.74 0.068 10.8 24.4

‘Data given are for a typical purification run using 20 rat pineal glands and an elution protocol as given in Figure 1.

curve of eluted enzyme activity was not symmetrical, although the curve of eluted protein was, suggesting an inactivation or denaturation of HIOMT exposed to low pH during elution. Less rigorous elution conditions (PH 4) eluted little activity.

Analysis of the acid eluate by SDS-PAGE showed a large enrichment of a protein of Mr - 38,000 in those fractions containing enzyme activity (Figs. 2 , 3 ) . Minor amounts of some proteins of larger molecular mass were present in all fractions, as revealed by silver staining and densitometry scanning. In two separate experiments, gels were stained with Coomassie blue, the strip of gel containing the prominent protein band at M, - 38,000 was cut out, and the protein was electroeluted. The electroeluted protein was subjected to hydrolysis (concentrated HC1 or methane sulfonic acid) and amino acid analysis, and the number of residues of each amino acid per mole of protein was calculated (Table 2). For many amino acids, the number of residues is

392 Sugden, Voisin, and Klein

“z

90 80 70 60 50 6-

2 40

$ 30 I

X

20

- - - -

- Ovalbumin

-

-

Lysozyme

1 0.2 0.4 0.6 0.8 1.0

Rf

Fig. 2. Estimation of molecular weight of HIOMT. Aliquots (- 5 pg of protein) of the HIOMT sample eluted by acid (see Fig. 1) were precipitated with ice-cold trichloroacetic acid (10% w/ v), the precipitate was washed 2 times with acetone, then dissolved in electrophoresis sample buffer (2.3% SDS, 5 % 0-mercaptoethanol, 10% glycerol, 60 mM Tris-HCI, pH 6.8). Standard proteins of known molecular weight (Bio-Rad) were run concurrently. Gels were stained with silver using a modification of the technique of Morrissey [19Sl]. The data shown are represen- tative of several batches of purified enzyme.

A MW

( x 10-3)

92 - 66 -

45 -

31 -

a h

Relative Optical Density

Fig. 3 . SDS-Polyacrylamide gel electrophoresis of AdoHcy-agarose purified HIOMT. “A” is a lane with a sample of the eluate containing the peak of enzyme activity (- 3 pg of protein) eluted by acid as in Figure 1, stained with silver. The negative image of lane A was scanned using a soft laser scanning densitometer (LKB, Gaithersburg, MD). Migration distances of protein standards (see Fig. 2) in this gel are indicated with their molecular mass. The sharp peak at the bottom of the densitometric trace indicates the migration front.

Purification of Rat HIOMT 393

TABLE 2. A m i n o Ac id Compos i t ion of Hydroxyindole-0-Methyltransferase

Assumed No. of residues Bovine’

Amino acid Rat‘ A B

Aspartic acid 31 22 26 Threonine 13 14 15 Seriiie 20 22 24 Glutamic acid 36 37 40 Proline 16 14 14 Glycine 31 33 35 Alanine 21 37 38 Valine 23 23 25 Methionine 5 4 4 Isoleucine 14 11 10 Leucine 27 48 52 Tyrosine 9 14 15 Phenylalanine 10 14 15 Histidine 6 7 6 Lysine 18 10 10 Arginine 12 28 26 Tryptophan 7 2 3-4 Cysteic acid -1 9 10

‘A, taken from Kuwano and Takahashi [1978]; B, taken from Jackson and Lovenberg [1971]. ’Rat HIOMT purified by affinity chromatography was run on 10% SDS-PAGE. The gel was stained with Coomassie blue and the major band at - 38 K daltons cut out and electroeluted overnight (see “Materials and Methods”). Residues given are the mean of duplicate analyses utilizing methane sulfonic acid (4 N) or concentrated HCI (6 N) hydrolysis of approximately 10 pmole of protein.

very similar to that reported for bovine HIOMT Uackson and Lovenberg, 1971; Kuwano and Takahashi, 19781. However, rat HIOMT is richer in aspartic acid, while the bovine enzyme is richer in basic amino acids (lysine + arginine). Furthermore, there are fewer hydrophobic amino acids (leucine + alanine) in the rat enzyme.

DISCUSSION

An AdoHcy-linked agarose, similar to that described originally by Kim et al. [ 19781, has been used in the present study to purify HIOMT from small quantities of rat pineal tissue. Similar affinity chromatography procedures have been included in the purification of protein carboxyl O-methyltransfer- ase [Kim et al., 1978; McFadden et al., 19831, phenylethanolamine N-meth- yltransferase [Hurst et al., 19831, and histamine N-methyltransferase [Matuszewska and Borchardt, 19831. Although each of these enzymes binds readily to AdoHcy-agarose, the conditions necessary for elution vary mark- edly [Kim et al., 1978; Hurst et al., 1983; Matuszewska and Borchardt, 1983; McFadden et al., 19831. This may relate to differences in the binding of each of these enzymes to AdoHcy [Borchardt and Wu, 19761 or to the exact nature of the linkage between AdoHcy and agarose in the particular gel used [Kim

394 Sugden, Voisin, and Klein

et al., 1978; Hurst et al., 19831. Nevertheless, rat HIOMT binds tightly to AdoHcy-agarose since AdoMet (up to 1 mM) was unable to elute the enzyme. High salt (1 M NaC1) eluted some activity, probably that bound to the column by ionic interactions. We were able to make use of the tight binding of enzyme activity and elute virtually all the other proteins bound to the column by washing with high salt followed by AdoMet to elute other pineal methyltransferases that may have bound to the column [Axelrod et al., 19611. HIOMT was then eluted by reducing the pH. The harsh elution conditions required reduced the recovery of enzyme activity considerably. However, the proteins eluted by acid (- 1 % of the total loaded) were highly enriched in a band at -38kD; an indentical molecular mass has been reported for chicken [Nakane et al., 19831 and bovine HIOMT Uackson and Lovenberg, 1971; Kuwano and Takahashi, 19781. Amino acid analysis of the 38-Kd band gave a composition very similar to bovine HIOMT in many respects, but suggested that rat HIOMT should be less hydrophobic than the bovine en- zyme and should have a more acidic PI.

Although recovery of enzyme activity using this method is limited, it appears that microgram quantities of highly purified HIOMT protein can be recovered. As little as 10 mg of pineal tissue (10 glands) and as much as 400 mg (400 glands) has been used successfully as the enzyme source. Thus, this method offers a rapid, simple procedure applicable to small amounts of material, which may be of particular value in attempts to purify HIOMT in sufficient quantity to permit the elucidation of the amino acid sequence and studies of the molecular characteristics of the active site.

ACKNOWLEDGEMENTS

We are grateful to Dr. Harry Chen (NICHD) for the amino acid analyses of purified rat HIOMT.

LITERATURE CITED

Axelrod, J . , H. Weissbach (1960) Purification and properties of hydroxyindole-0-methyltrans- ferase. J. Biol. Chem. 236:211-213.

Axelrod, J . , P.D.Maclean, R.W. Albers, H. Weissbach (1961) Regional distribution of methyl- transferase enzymes in the nervous system and glandular tissues. In: Regional Neuro- chemistry. S.S.Kety and J. Elkes, eds., Pergamon Press, New York, pp. 307-311.

Borchardt, R.T., Y.S. Wu (1976) Potential inhibitors of S-adenosylmethionine-dependent meth- yltransferase 5 . Role of the asymmetric sulfonium pole in the enzymatic binding of S- adenosyl-L-methionine. J. Med. Chem. 19: 1099-1103.

Huiikapiller, M.W., E. Lujan, F. Ostrander, L.E. Hood (1983) Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods En- zymol. 91:227-236.

Hurst, J.H., R.B. Guchhait, M.L. Billingsley, J.M. Stolk, W. Lovenberg (1983) Phenylethanol- amine N-methyltransferase: Notes on its purification from bovine adrenal medulta and separation from protein carboxy-methyltransferase. Biochem. Biophys. Res. Commun.

Jackson, R.L., W. Lovenberg (197 1) Isolation and characterization of multiple forms of hydroxy- indole-0-methyltransferase. J. Biol. Chem. 246:4280-4285.

Kim, S., S. Nochumson, W. Chin, W.K. Paik (1978) A rapid method for the purification of S- adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography. Anal. Biochem. 8 4 :4 15-422.

11 2 : 106 1- 1068.

Purification of Rat HIOMT 395

Kuwano, R. , Y. Takahashi (1978) Purification of bovine pineal hydroxyindole-0-methyltrans- ferase by immunoadsorption chromatography. J. Neurochem. 31 2315-824.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

Matuszewska, B., R.T. Borchardt (1983) Guinea-pig brain histamine N-methyltransferase: Pu- rification and partial characterization. J. Neurochem. 41: 113-118.

McFadden, P.N., J. Horwitz, S. Clarke (1983) Protein carboxyl methyltransferase from cow eye lens. Biochem. Biophys. Res. Commun. 113:418-424.

Morrissey, J.H. (1981 ) Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310.

Nakane, M., E. Yokoyama, T. Deguchi (1983) Species heterogeneity of pineal hydroxyindole- 0-methyltransferase. J. Neurochem. 40:790-796.

Sugden, D. , D .C. Klein (1983) Adrenergic stimulation of rat pineal hydroxyindole-0-methyl- transferase. Brain Res. 265:348-351.

Sugden, D., M.A.A. Namboodiri, J.L. Weller, D.C. Klein (1983) Melatonin synthesizing en- zymes: Serotonin N-acetyltransferase and hydroxyindole-0-methyltransferase. In: Meth- ods in Biogenic Amine Research. S. Parvez, T. Nagatsu, I. Nagatsu, and H. Parvez, eds., Elsevier/North-Holland Biomedical Press, Amsterdam, Chapter 25, pp. 567-572.