A Salsolinol Synthase from Rat Brain

Biologia 66/6: 1183—1188, 2011 Section Cellular and Molecular Biology DOI: 10.2478/s11756-011-0134-y Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain Xuechai Chen, Abida Arshad,HongQing,RuiWang, Jianqing Lu & Yulin Deng* School of Life Science & Technology, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 100081, People’s Republic of China; e-mail: [email protected] Abstract: Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra. Key words: salsolinol; dopamine; salsolinol synthase; enzyme activity; protein purification. Abbreviations: AcH, acetaldehyde; DA, dopamine hydrochloride; ECD, electrochemical detection; EDTA, ethylene diamine tetraacetic acid; PD, Parkinson’s disease; Sal, Salsolinol – 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; (R)-Sal, (R)-enantiomer salsolinol; (S)-Sal, (S)-enantiomer salsolinol; SD, Sprague-Dawley; THP, tetrahydropapaveroline. Introduction However, the transport of Sal into brain through the blood-brain barrier is excluded (Origitato et al. 1981; The pathogenesis of idiopathic Parkinson’s disease Song et al. 2006), indicating the in situ synthesis of Sal (PD) remains to be elucidated. Many reports have in the brain. Sal has an asymmetric centre at C1 and revealed the involvement of neurotoxins in the de- exists as (R)- and (S)-enantiomers (Deng et al. 1997). terioration of nigrostriatal dopamine system in PD In humans, reports have shown that (R)-enantiomer of (Maruyama et al. 1997). Three endogenous alkaloids Sal can only be detected (Deng et al. 1995), whereas were proposed as potential candidates of dopaminergic others indicated that the enantiomer ratio of (R)-/(S)- neurotoxins named salsolinol (l-methyl-6,7-dihydroxy- Sal was approximately 2 (Musshoff et al. 2000). In pre- l,2,3,4-tetrahydroisoquinoline; Sal), tetrahydropapave- vious studies, the concentration of (R)-Sal was found roline (THP) derivative (1BnTIQ) and β-carbolines. to be higher than the (S)-Sal, depicting the presence Sal, in particular, is toxic to dopaminergic neurons of such biosynthetic enzyme in etiology of disease. Fig- and has recently gained much attention due to its ure 1 illustrates two proposed synthetic pathways of structural similarity with 1-methyl-4-phenyl-1,2,3,6- Sal from dopamine and acetaldehyde (AcH). The first tetrahydropyridine, a potent neurotoxin that specifi- one produces the racemic mixture of both enantiomers cally degenerates the nigrostriatal dopaminergic neu- (Robbins et al. 1968; Zhu et al. 2008), the latter pro- rons, resulting in dopamine deficiency in the striatum, duces (R)-Sal only (Naoi et al. 1996; Rojkovicova et al. and hence the symptoms of Parkinsonism (Quan et al. 2008). Accordingly, the surplus of (R)-Sal detected in 2005). Therefore, Sal may have an influential role di- the human brain may be biosynthesized by this putative rectly or indirectly in causing PD. enzyme. Numerous publications have proposed the exis- Sal is a kind of catechol isoquinoline that can be tence of (R)-salsolinol synthase, however, to our knowl- found in brain, cerebrospinal fluid, blood, and urine edge, only one paper has reported the primary purifi- (Haber et al. 1996). It can also be detected in some cation of the enzyme from human brains, published by foods and beverages, such as dried banana and port Naoi et al. (1996) from Japan. From then on, salsolinol wine, suggesting that consumption of such daily food synthase has not been extensively purified and charac- items can affect Sal level in tissues (Smythe et al. 1985). terized, and it has also not been reported whether this * Corresponding author c 2011 Institute of Molecular Biology, Slovak Academy of Sciences 1184 X. Chen et al. Fig. 1. Synthetic pathways of Sal from dopamine and acetaldehyde. Pathway A is non-enzymatic Pictet-Spengler reaction; it produces racemic mixture of both (R)- and (S)-Sal. Pathway B is catalyzed by (R)-salsolinol synthase, producing only (R)-Sal. enzyme exists in the other animal brains or not. Hence adjusted to pH 7.4 with 1 M HCl. Following centrifugation ◦ further research in this field is necessary to explore the at 100,000×g for 1 h at 4 C, the supernatant was collected mechanism of action and possible role of (R)-salsolinol as crude enzyme. Crude enzyme fraction was incubated in synthase in the pathogenesis of PD. 50 mM Tris-HCl (pH 7.4) with 1 mM DA and 1 mM AcH in a total volume of 1 mL (Naoi et al. 1996; De-Eknamkul In this study we conducted a series of experiments ◦ et al. 1997). After incubation at 37 C for 30 min, 20 µLof to examine the existence of salsolinol synthase in rat 1 M perchloric acid containing 1 mM EDTA and sodium brain. Chiral analysis was done to demonstrate the fact metabisulphite was added to stop the reaction. After mix- that this enzyme has an enantio-selectivity. Moreover, ing and centrifugation at 17,000×g for 15 min, the sample the distribution of this enzyme in various regions of was diluted 25 fold and filtered through a 0.22 µm cellulose brain was thoroughly investigated. membranes before storing at −80 ◦C for further experimen- tations. Material and methods HPLC-electrochemical detection apparatus and chromato- Chemicals and substrates graphic conditions Dopamine hydrochloride (DA), AcH, Sal, sodium 1-hepta- Enzyme activity was assayed using HPLC-electrochemical nesulphonate and HPLC-grade methanol were purchased detection (ECD) (ESA, Chelmsford, MA, USA) as described from Fisher Scientific Canada (Edmonton, Canada). Tris- previously (Maruyama et al. 1996). Briefly, the oxidation HCl, perchloric acid, ethylene diamine tetraacetic acid voltage of a model 5020 guard cell was 400 mV, 350 mV, (EDTA), sodium metabisulphite, citric acid and pentobar- 100 mV and −50 mV. Samples were separated on a reverse- bital sodium were purchased from Sigma. Disodium EDTA phased Inertosil ODS-C18 column (4.6 (i.d.) × 150 mm), was purchased from Sino-American Biotechnology Com- with a mobile phase (pH 4.0) of citric acid (40 mM), pany. All other chemicals were obtained from the Beijing Na2HPO4 (20 mM), Na2EDTA (0.3 mM), 5% methanol, Chemical reagent Company in China and all of the chemi- and 1.0 mM heptane sulphonic acid at a flow rate of 1.0 cals used were of analytical grade and organic solvents were mL/min. The quantity of Sal was calculated using a stan- of HPLC grade. A Milli-Q water purifying system was uti- dard curve generated by standard substances. The salsolinol lized to generate 18.2 MΩ deionized water. synthase activity was demonstrated by the increment of Sal during per min per mg of protein at 37 ◦C, and is represented −1 −1 Brain dissection as nmol min mg . The protein was quantified using the Sprague-Dawley (SD) rats, weighing 200 ± 20 g, were ob- Bradford method (Bradford 1976). tained from Central Animal House of Chinese Academy of Medical Sciences. Rats were kept under standard laboratory Preliminary purification of salsolinol synthase conditions, at room temperatures and were supplied with The crude enzyme fraction was used for preliminary purifi- food and water. The rats were anesthetized with sodium cation. The sample was first presented on a PA-DEAE-02 pentobarbital (64.8 mg/mL, 50 mg/kg) and decapitated (ac- column (20 (i.d.) × 100 mm) at the rate of 3.0 mL/min. A cording to institutional protocols). The brains were removed non-linear gradient elution of eluents A (25 mM Tris-HCl by standard autopsy procedures and cortex, striantum, sub- buffer solution and 1 M NaCl solution) and B (25 mM Tris- stantia nigra, and cerebellum were quickly dissected on a HCl buffer solution, pH 7.4) was used for the separation. ◦ cold plate at 4 C for subsequent investigations (Musshoff The elution programmer was optimized and conducted as ◦ et al. 1999). All specimens were frozen at −80 Cuntilbio- follows: over the range from 0.01 min to 5.00 min, 0% A chemical analysis. was used, from 5 min to 25 min, 0–60% A was used, 60– 90% A was used in the range of 25 min to 45 min, and then Sample preparation 90–100% A was used from 45min to 48min, finally, sepa- Tissue samples (0.2–0.5 g) were weighed and transferred to ration was done for period from 48 min to 53.01 min with centrifugation tubes. The sample were homogenized with 100% A. Detection was accomplished via UV at 280 nm. In an Ultra Turrax in 1 mL of a solution of 50 mM Tris-HCl, total, 53 fractions were collected after every min. The same Salsolinol synthase from rat brain 1185 Fig. 2. Production of Sal detected by HPLC-ECD. (A) Chromatographic patterns: a: enzyme, b: DA+AcH, c: DA+enzyme, d: AcH+Enzyme, e: DA+AcH+enzyme.

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