0 1992 MUNKSGAARD

Determination of kinetic properties of serotonin-N-acetyl trans ferase in bovine pineal gland using HPLC with fluorimetric detection

Fajardo N, Abreu P, Alonso R. Determination of kinetic properties of serotonin-N-acetyltransferase in bovine pineal gland using HPLC with fluorimetric detection. J. Pineal Res. 1992: 1330-84.

Abstract: The determination of serotonin-N-acetyltransferase (NAT) activity in the bovine pineal gland and other rat tissues was based upon the separation and detection of N-acetyltryptamine formed from tryptamine and acetyl CoA by means of high performance liquid chromatography with fluorimetric detection. In the bovine pineal the enzyme exhibited a Km value of 31.45 t 4.98 pM and a Vmax value of 30.90 t 1.18 pmol N-acetyltryptamine/min/mg protein for tryptamine, and a Km value of 28.72 2 7.50 pM and a Vmax value of 25.90 t 1.50 pmol N-acetyltryptamine/min/mg protein for acetyl CoA. The present method is simple, allows the determination of NAT activity from a variety of enzyme sources, has application to pharmacological studies of NAT regulation in tissue cultures, and provides an alternative

I to current radioenzymatic assays.

Introduction

N-acetylation of pineal serotonin (5-HT) is cata- lyzed by the cytoplasmatic enzyme N-acetyltrans- ferase (NAT; E.C. 2.3.1.87) arylalkylarnine-N- acetyltransferase), which is considered to be rate limiting in the production of melatonin in most animal species [Reiter, 19911. With a few excep- tions, both the activity of NAT in the pineal gland, as well as the melatonin content, display daily rhythmicity, with highest values at night or during the dark phase of an imposed 1ight:dark cycle [Klein and Weller, 1970; Rudeen et al., 1975; Alonso et al., 1980; Arendt, 19841. Although the endoge- nous substrate is 5-HT, most investigators have used tryptamine in the NAT assay since the enzyme exhibits a similar Km and Vmax for both com- pounds [Voisin et al., 19841 and N-acetylserotonin can be metabolized to melatonin, but not N-acetyl- tryptamine. Acetyl CoA, which is the acetyl donor for the N-acetylation of 5-HT, has been shown to act also as an important stabilizer of this enzymatic activity [Binkley et al., 19761. The lability of pineal NAT has precluded any in-depth investigation, and few kinetic determinations have been made [Degu- chi, 1975; Chan and Ebadi, 1981; Chan and Ebadi, 1980; Morton, 1986; Reiter et al., 19901. The present study was undertaken in order to develop a simple chromatographic assay for NAT from dif-

Natalia Fajardo, Pedro Abreu, and Rafael Alonso Laboratorio de Neuroendocrinologia, Departa- mento de Fisiologia, Facultad de Medicina. Universidad de La Laguna, Tenerife, Spain

Key words: NATGnzyme kinetics-pineal gland-HPLC

Address reprint requests to Rafael Alonso, Laboratorio de Neuroendocrinologia, Departmen de Fisiologia, Facultad de Medicina, Universidad de La Laguna, Apartado 55. La Cuesta, 38320, Tenerife, Spain

Received January 24, 1992; accepted June 12, 1992.

ferent tissues, and, in addition, to elucidate the catalytic behavior of the enzyme. This information may serve to increase our understanding of pineal biochemistry and will allow a more accurate inter- pretation of any physiological or pharmacological effect exerted on the enzyme. We have measured the activity of NAT and characterized its kinetic properties in the bovine pineal gland. In addition, we have applied this enzyme assay to other rat tissues that also synthesize melatonin, including the pineal, retina, and Harderian gland.

Materials and methods

Preparation of tissue

Bovine pineal glands were dissected from fresh brains obtained at a local slaughterhouse; they were frozen immediately on solid CO,. Pineal glands, retinas , and Harderian glands were obtained from male Sprague-Dawley rats sacrificed at either 1400 or 0200 (lights on, 0800; lights off, 2000). Each bovine pineal and the two Harderian glands were homogenized with a polytron homogenizer (Ultra- Turrax T25, at 8,000 rpm) for 20 sec in 2 ml of 0.25 M sucrose. Individual rat pineals and the two retinas were homogenized by sonication (100 W, 5 sec) in 200 p1 or 300 p1 of 0.25 M sucrose, respectively. After centrifugation at 5,400g for 5 min, superna-

80

Kinetic properties of NAT in pineal gland

tants were individually frozen and stored at - 80°C until assayed.

Culture techniques

Rat pineal glands were cultured using a method described previously by Brammer et al. [ 198 11 with some modifications. Individual pineals were im- mersed in 1 ml of pH 7.4 culture medium (Falcon organ culture dish 3037, Falcon Plastics, Los An- geles, CA), and then incubated at 37°C under 95% air/5% CO,. The culture medium (BGJb Fitton- Jackson modification, Grand Island Biological Co., Grand Island, N.Y .) was supplemented with ascor- bic acid (0.1 mg/ml), glutamine (0.2 mM), BSA fraction V ( 1 mg/ml), penicillin (100 U/ml) and streptomycin ( 100 pg/ml), and preincubated for 30 min before sampling. Isoproterenol (ISO, 10 pM) was dissolved in water and added in a volume that did not exceed 1% of the incubation volume. After incubation for 5 hr, pineal glands were removed from the medium and frozen at -80°C until as- sayed.

Assay of NAT activity

NAT activity was assayed by measuring the amount of N-acetyltryptamine formed from acetyl CoA and tryptamine by high performance liquid chromatog- raphy (HPLC) with fluorimetric detection [Bradford et al., 19901. Aliquots of SO pl homogenate were mixed with 25 p1 of 8 mM tryptamine and 25 pl of 4 mM acetyl CoA (Sigma Chemical Co., Poole, U.K.) in 0.1 M phosphate buffer, pH 6.5, contain- ing 5 Mm EGTA. Samples were incubated at 37°C while shaking for 40 min, and the enzymatic reaction was stopped by addition of I ml of ice-cold to1uene:acidified isoamyl alcohol (99: 1 , vol/vol; isoamyl alcohol was acidified with 1 M HCl, 3: 1, volivol). After shaking for 5 min, samples were centrifuged at 1 1,000 rpm for 5 min and aliquots of 0.5 ml from the organic phase were taken to dryness (Speedvac Concentrator, Savant Instruments, Inc., Farmingdale, NY, U.S.A.). The residue was redis-

p1 were injected into the chromatographic system. The protein content was determined by the method of Bradford [ 19761 using bovine serum albumin as a standard.

In order to carry out the kinetic studies, the final concentration of acetyl CoA was varied up to 5.0 mM, while keeping the concentration of tryptamine at a constant level of 8 mM. Conversely, in a different series of experiments, the final concentra- tion of tryptamine varied up to 5 mM, while keeping the concentration of acetyl CoA at a constant level of 4 a.

solved in 100 pl of mobile phase, and aliquots of 25

Chromatographic procedure

Aliquots prepared as described above were injected into a C- 18 Spherisorb ODS reversed-phase column (particle size 5 pm, Scharlau S.A.; Barcelona, Spain). The mobile phase consisted of 0.1 M acetic acid, 0.1 M sodium acetate, 50 mg/l EDTA and 15% acetonitrile (vol/vol) in distilled deionized water (Millipore Q System, Millipore Iberica S.A., Barcelona, Spain), which was adjusted to pH 5.2 and filtered and degassed before used. The system was run at a flow rate of 1.0 mlimin (Waters 510 pump, Millipore Iberica, Barcelona, Spain). The fluorescence detector (LS 40, Perkin Elmer Ltd, Buckinghamshire, U.K.) was set at 285 nm and 360 nm as excitation and emission wavelengths, respec- tively. The identification of peaks by retention time and quantification of N-acetyltryptamine by peak height was done using a HP 3396A integrator (Hewlett Packard Co., Avondale, U.S.A.).

Data analysis

The kinetic analysis of NAT activity was performed by using a nonlinear regression data analysis pro- gram (Enzfytter) [Leatherbarrow, 19871. In addi- tion, the kinetic parameters were contrasted by means of linear regression analysis using Hanes’ equation [Hanes, 19321. Data are expressed as means 5 SEM, and comparison between pairs of means was performed by Student’s t-test.

Results and discussion

The first method for the determination of NAT activity in pineal gland was based on the measure- ment of 14C-N-acetyltryptamine formed by the transfer of a I4C-acetyl group from I4C-acetyl CoA to tryptamine [Deguchi and Axelrod, 19721. The introduction of a chromatographic procedure was made by Bradford et al. [I9901 including, in the same report, the chemical synthesis of N-acetyl- tryptamine. In our case, we have avoided the use of ion-pair chromatography since reverse phase chro-

N-acetyltryptamine peak and allows the addition of a smaller quantity of organic modifier. This method has proved to be very useful for assaying low levels of NAT activity from a variety of enzyme sources (pineal gland, Harderian gland, and retina) since the coupling of HPLC separation with fluorimetric detection has led to a great improvement in both sensitivity and specificity for N-acetyltryptamine. Under the conditions described above, N-acetyl- tryptamine displayed a retention time of 22.8 min, which resulted in a clear baseline separation from the substrate tryptamine and other compounds

matography is sufficient for the resolution of

81

Fajardo et al.

A

- I

0 10 20 30 40 60 60 7 0 10 90

0 Y

TIME(minutes)

present in the reaction mixture (Fig. 1). Linearityof detector response was determined by injecting dif- ferent amounts of authentic N-acetyltryptamine (0.5, 1 .O, 2.5, 5.0, 10.0, and 20.0 ng) and running them in duplicate. The correlation coefficient was 0.988 and the overall N-acetyltryptamine recovery for the extraction and estimation process was 93- 97%.

The rate of N-acetyltryptamine formed was linear up to 80 min (Fig. 2A). On the other hand, N-acetyltryptamine formation also increased in a linear fashion with amounts of tissue added to the incubation medium up to 500 pg/tube (Fig. 2B), thus indicating that the reaction rate is proportional to the amount of enzyme in the system. This allows the investigator to work with a wide range of incubation times, as well as permitting the use of different tissues having various protein contents.

The kinetic analysis revealed a Km value of 31.45 -+ 4.98 pM and a Vmax of 30.9 -t 1.18 pmol of N-acetyltryptamine/min/mg protein for tryptamine (Fig. 3A), and a Km value of 28.72 -+ 7.5 p M and a Vmax of 25.90 k 1.5 pmol N-acetyltryptamine/min/mg protein for acetyl CoA (Fig. 3B); this is consistent with previous data using crude enzyme preparation from bovine pineal gland [Chan and Ebadi, 19801, and also indicates a similar enzyme affinity for both tryptamine and acetyl CoA. It has been recently shown that addition of EGTA causes a significant increase of NAT activity in rat and chicken pineal glands [Rodriguez-Cabello et al., 19901, which has been attributed to a

d c I o $00 200 aoo 400 600

- f ig protein/tube

0 E a Y

Fig . 2 . NAT activity in bovine pineal gland as a function of either (A) incubation time or (B) protein concentration. In (A), each tube contained 114.8 pg of protein, while in (B) the incubation time was fixed at 40 min. The concentrations of tryptamine and acetyl CoA were fixed at 8 mM and 4 mM, respectively, and the assay was performed in the presence of 5 mM EGTA. NAT activity is expressed in pmol of N-acetyltryptamine per tube.

Kinetic properties of NAT in pineal gland

32-

24-

A W

0 8

I ‘ 1 . 1 ’ 1 0 1 3 4 5

Tryptamine (mM)

B & I

0

--I 0

0

Acetyl-CoA (mM)

Michaelis-Menten plots of NAT activity in bovine pineal gland. These studies were carried out while (A) varying the concentration of tryptamine from 0.003125 to 5.0 mM, with a fixed concentration of 4 mM acetyl CoA or (B), varying the concentration of acetyl CoA from 0.003125 to 5.0 mM, with a fixed concentration of 8 mM tryptamine. The initial velocity (NAT activity) was expressed as pmol N-acetyltryptaminelminlmg protein. Each point represents the mean of four determinations. The insets show the linear transformation obtained by rearranging the kinetic equation according to Hanes [1932]. Straight line slope and intercept (X axis) are 1/Vmax and -Km, respectively.

protective effect on the enzyme during the incuba- tion process, suggesting that NAT enzyme could be sensitive to proteases/peptidases metal dependent. In the present work, the addition of EGTA produced a slight enhancement of enzyme activity starting at

A a concentration of 5 mM, which, therefore, was 0 chosen as the optimum to measure NAT activity in

bovine pineal gland (Fig. 4). h 1s

Under the conditions described, the method has a \ k! detection limit of 0.5 pmol N-acetyltryptamine/

min/mg protein, and it is therefore useful for \ measuring NAT activity in different tissues, even Y 5 *E n E lolfl--I during obtained the with light this period method (Table are 1 ) . in Also, good agreement the values

with those reported previously (Table 2). In sum- mary, the present protocol is suitable for routine enzyme analysis and kinetic studies, and reduces both sample manipulation required in the classic radiochemical procedure as well as assay cost. In addition, the improved assay also permits its appli-

0 S 10 2o 25 cation to biochemical and pharmacological studies on NAT regulation in several melatonin-forming tissues.

Acknowledgments

c

C

._ > 0-

c 0 6

c o z si EQTA Concentration (mM)

Fig . 4. Effect of EGTA concentration on NAT activity in bovine pineal gland. Samples were incubated for 40 min at 37°C as described in “Materials and methods” in the presence of different concentrations of EGTA. Data are expressed as mean * SEM of four determinations.

This work was supported by Spanish DGICYT PM88-0206. The authors wish to thank Juan Plata, Veterinary Chief of La Laguna Town Hall, for supplying bovine pineal glands, and Dr. G. Hernandez for his technical assistance.

83

Fajardo et al.

TABLE 1. Values of NAT activity from different melatonin-forming tissues of the rata

Light Dark

Cultured pineal gland Control 10.44 c 0.63 IS0 (1 k M ) 58.74 t 2.73

Pineal gland 4.76 t 0.65 241 .OO * 49.20** Harderian gland 125.50 c 5.00 161 .OO f 6.00* Retina 28.60 f 1.60 26.80 t 1.80

aNAT activity was measured in pineals, retinas, and Harderian glands of male rats obtained during light (1400) or dark periods (0200) (lights on, 0800; lights off, 2000). In culture experiments, rat pineal glands were harvested 5 hr after addition of lsoproterenol (IS0 1 kM) or vehicle. All tissue samples were isolated, frozen, and assayed for NAT activity as described in “Materials and methods.” Number of samples = five. NAT activity is expressed as pmol N-acetyltryptamine/min/gland (pineal gland) or as prnol N-acetyltryptaminelrnin/mg protein (retina and Harderian gland). * P < 0.01; * * P < 0.001.

TABLE 2. Typical values of NAT activity in rat pineal gland according to different assay proceduresa

Method Reference NAT activity

Light Dark

REAb Rudeen et al. [I9753 8.0 t 0.4 133.3 c 16.6 REA Binkley, S.A. [I9831 6.6 t 0.3 183.4 t 12.5 REA Champney et al. [I9841 5.0 k 0.2 116.7 c 8.3 HPLCC Bradford et al. [I9901 4.2 t 0.3 300.0 t 56.0 HPLC This method 4.8 t 0.6 241.0 c 49.2

aData are expressed as mean t SEM calculated from the individual values given in the original report. NAT activity is expressed as pmol N-acetyltryptamine/min/gland. bREA: Radioenzymatic Assay. ‘HPLC: High performance liquid chromatography.

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