Uptake of tyramine by rat hepatocytes

Uptake of tyramine by rat hepatocytes

Biochimica et Biophysica Acta, 1176 (1993) 77-82 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00 77 BBAMCR 13347 Up...

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Biochimica et Biophysica Acta, 1176 (1993) 77-82 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00


BBAMCR 13347

Uptake of tyramine by rat hepatocytes Zhi-Duan Zhong, Simone Wattiaux-De Coninck and Robert Wattiaux Laboratoire de Chimie Physiologique, Facultes Universitaires, Notre-Dame de la Paix, Namur (Belgium)

(Received 30 July 1992)

Key words: Tyramine; Transporter; Hepatocyte; (Rat)

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a K m of 39 /~M and a Vmax of 270 pmol/min/105 cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3, SCN- and SO4z , which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.

Introduction Tyramine, a sympathomimetic amine is found in small amounts in chromaffin granules and sympathetic nervous tissues [1]. Its physiological function is still unknown. An excess of tyramine in general circulation can induce peripheral vasoconstriction by causing a release of catecholamines from sympathetic nerve endings and adrenal medulla. Such a situation does not normally take place, although this amine is relatively abundant in some foods (cheese, chocolate) and is probably produced by bacterial degradation in the intestine. This suggests that tyramine could be quickly removed by the liver before it enters general circulation. However, in some circumstances, for example after intake of monoamine oxidase inhibitors and in certain nutritional conditions, the concentration of tyramine in blood may increase and cause a severe hypertensive crisis [2]. Patients who suffer from mi-

Correspondence to: R. Wattiaux, Laboratoire de Chimie Physiologique, Facultes Universitaires Notre-Dame de la Paix, 61, rue de Bruxelles, B-5000 Namur, Belgium. Abbreviations: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; NEM, N-ethylmaleimide; pAPMA, p-aminophenylmercuric acetate; EMTSA, ethylmercurithiosalicyclic acid; PITC, phenylisothiocyanate; pCMB, p-chloromercuribenzoate; PMC, phenylmercuric chloride; pCMPS, p-chloromercuriphenylsulfonate; EAI, ethylacetimidate; TNBS, trinitrobensulfonic acid; PGO, phenylglyoxal; NBS, n-bromosuccinimide; DEP, diethylpyrocarbonate.

graine are generally advised not to eat cheese, since the tyramine it contains could induce a migraine attack [31. To our knowledge, detailed information concerning the uptake of tyramine by the liver is not available. In the work reported here, we have studied this uptake mainly by using isolated hepatocytes. Our results show that tyramine can be quickly taken up by hepatocytes, both by simple diffusion and a transporter-mediated process. Materials and Methods The experiments were performed on male Wistar rats, weighing about 200 g, that were starved for 20 h before killing. Hepatocytes were isolated according to the method of Seglen [4] with slight modification: both Ca2+-free and collagenase perfusion were carried out at 34°C for 6 min. For all the experiments, only the preparations where more than 90% of the cells excluded Trypan blue were used. After isolation, the cells were kept at 4°C until being used. We used the cells no more than 2 h after being isolated. In that condition, the quality of the cells did not change. Unless stated in the legends of the figures, transport assays were performed in R P M I 1640 medium without serum, containing 10 m M H e p e s (pH 7.6), at 37°C. 15 s incubation time was chosen. In this case, the amounts of [14C]tyramine within the cells after incubation did not exceed 5% of the amount added to the medium. In

78 brief, 3 • 105 cells were preincubated at 37°C for 2 min. They were then incubated with 6.6 tzM [~4C]tyramine (spec. act. 2.07 GBq/mmol) in a final volume of 200/xl in the absence or presence of inhibitors (which were added simultanously with the labelled tyramine, except for those indicated in the legends). The uptake reaction was stopped by the addition of 400 Izl ice-cold medium. Following this, 200-/21 samples were immediately laid in an Eppendorf microtube, at the top of 150 t~l oil (a mixture of dibutylphthlate and dioctylphthlate of 1.20 g / m l density) and centrifuged in an Eppendorf centrifuge at 10000 × g for 30 s. The cell's pellet was cut and added to 0.5 ml boiling SDS solution (0.1%) in a scintillation counting vial. After 5 min., 4 ml of a liquid-scintillation cocktail (Aqualuma Lumac, Landgraaf, The Netherlands) was added. Counting was performed in a Beckman/3 counter. In the cases in which the effects of amino acids and their modifiers were tested, experiments were carried out with perfusion buffer which was composed of NaCI (mM): 137, KC1 5.4, Na2HPO 4 • 2H20 3.4, K H 2 P O 4 3.5, M g S O 4 " 7H20 8.1, NaHCO 3 24.4 and CaCI 2 • 2H20 2.0 (pH 7.6). The extracellular water contamination was measured by adding [14C]sucrose to hepatocyte suspension before centrifugation and determining the amount of radioactivity in the cell pellet. Less than 0.13% of the added radioactivity was present in the pellet. So, the contamination with extracellular water is always largely lower (2.02 + 0.10% (n = 3) of the total radioactivity associated with the cell pellet in [14C]tyramine uptake experiments). Since simple diffusion and transporter-mediated processes coexist in the uptake of tyramine by hepatocytes, the transporter-mediated uptake and ICs0 (the concentration of substance which causes half inhibition of tyramine uptake) was calculated after eliminating the simple diffusion part which accounts for about 25% of the total amount of tyramine taken up by hepatocytes under our experimental conditions. For this reason, tyramine transport mentioned in figures and tables refers to transporter mediated tyramine uptake. The relative affinity was calculated according to the formula of Burgen and Iversen [5]: (IC50 of phenethylamine/Relative affinity = ICs0 of substance) × 100%. All the chemicals were purchased from standard sources. Results

Uptake of tyramine by rat liver and isolated hepatocytes The main source of tyramine in general circulation comes from undeaminated tyramine after its passage through intestine and liver. To determine the proportion of tyramine taken up by the liver after its intestinal absorption, the [~4C]tyramine present in that organ was measured at various time after portal vein injection.









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0 35

Time ( rain ) Fig. 1. Uptake of [14C]tyramine by rat liver (A) and isolated hepatocytes (B). (A) Rat liver was perfused for 3 min with ice-cold 0.9% NaCI at various times after injection of [14C]tyramine (3 nmol) to the portal vein. 50 /zl homogenised liver (diluted 4 times with 0.25 M sucrose) was mixed with 4 ml liquid-scintillation cocktail (Aqualuma Lumac, Landgraaf, The Netherlands) before being counted in a Beckman /3 counter. Results are given as percentages of the amount injected to the rat. (B) Freshly isolated rat hepatocytes were incubated with [14C]tyramine (6.6/zM) at 37°C in RPMI 1640 medium. At the indicated times, 66 /zl mixture containing 105 cells were sampled and laid in an Eppendorf microtube, on the surface of 150 /zl oil (a mixture of dibutylphthalate and dioctylphthalate of 1.20 g / ml density) and centrifuged in an Eppendorf centrifuge for 30 s. The treatment of cell's pellet and the radioactivity counting are described in Materials and Methods. The total volume of hepatocytes was estimated according to the data given by Loud [11]. The results are given as either [14C]tyramine (pmoi/mg proteins) ([]) or the ratio of the radioactivity concentration inside (C i) and outside (C o) of the cells ( • ).

Fig. 1A shows that 20 s after injection, 57% of the [14C]tyramine is associated with the liver. Then, the radioactivity quickly decreases with time. Fig 1B illustrates the tyramine uptake by isolated hepatocytes. Five min after adding [14C]tyramine to the cell suspension, the concentration of tyramine within the cells is 17-times higher than that found in the outside medium. After that, its intracellular concentrations quickly diminished. This decrease probably originates from the catabolism of the amine particulary by monoamine oxidase A and B [6] and the fact that the concentration of [14C]tyramine in the medium decreases with time. In fact, we have observed (not shown) that in the presence of chloroquine, an inhibitor of monoamine oxidase, cell radioactivity reaches a higher peak and subsequently, decreases more slowly. However, during the first minute of incubation, the curves with and without chloroquine coincide.






Effects o f oligomycin, azide, cyanide. 2.4-dinitrophenol and FCCP on the tyramine uptake



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3.106 hepatocytes in 2 ml PRMI 1640 culture medium (pH 7.6) were preincubated with testing substances in the presence of 95% 0 2 + 5% CO 2 at 37°C before performing the transport measurement as described in Materials and Methods.

[] ~ ' ~

, i /



~ 6001. n

=~ 400-



Preincubation time (min) at 37°C

Tyramine transport (% of control)

Oligomycin 2,4-Dinitrophenol NaN 3 KCN FCCP

12.5/zM 1 mM 20 mM 20 mM 1 mM

20 20 40 40 2

50 32 68 54 1.3




O. 0




Fig. 2. Kinetic analysis of [14C]tyramine uptake by hepatocytes. The procedure for tyramine transport measurement is as described in Materials and Methods. The curve (a) fitting the experimental points ([]) can be resolved into a saturable process (c) with a Vmax of 270 pmol/min per 105 cells and a K m of 39 /zM as well as a linear uptake (b): v = 1.88.10 -6 [S].

a protonophore, inhibits only at high concentration (1 mM) the penetration of tyramine into hepatocytes (Table I). To investigate the influence of membrane potential on tyramine uptake, chloride was substituted in the perfusion medium by lipophilic anions (NO3, SCN-) and by a hydrophilic, less permeant anion: SO42-. As shown by Table II, in the presence of NO 3 and SCN-,

General properties of tyramine uptake in hepatocytes


Tyramine c o n c e n t r a t i o n


The influence of concentration on tyramine uptake is illustrated in Fig. 2. The curve is biphasic and can be resolved into a linear and a hyperbolic curve with a K m of 39 /zM and a Vm~x of 270 pmol/min/105 cells, suggesting that both simple diffusion and a transporter-mediated mechanism are involved in this uptake process. The carrier-mediated uptake is pH- and temperature-dependent.and requires an activation energy of 12.9 kcal/mol (Q10 = 2.0) (Fig. 3a,b). Adding Mg 2+ATP to the system does not stimulate tyramine transport (data not shown). In the presence of cyanide and azide, inhibitors of respiration, and oligomycin and dinitrophenol, respectively inhibitor and uncoupler of oxidative phosphorylation, the entry of tyramine across the membrane can be blockaded to some extent. FCCP,

The influences of N O 3 , S C N - and SO f - on the uptake o f tyramine

The isolated hepatocytes were pelleted and suspended in the perfusion buffer where NaC1 (137 mM) was presented or replaced by equal molar of NaNO3, NaSCN or Na2SO 4. With the respective buffer the cells were washed twice at 4°C and resuspended to 6.106 cells/2 ml. 3.105 cells were preincubated for 2 min at 37°C before the uptake measurements which were performed, in the appropriate buffer, according to the procedure described in Materials and Methods. * The differences are significant. Anion

Tyramine transport (% of control, ,g ± S.D., n = 3)

Cl-(control) NO; SCNSO~-

1 ~ ±1.8 83.6±1.3 76.8±5.4 2.5±0.3


< 0.01 * < 0.05 * < 0.001 *


B 9



12.9 Keal/tool 8





7 pH









6 3.7

[1/T] xlO00

Fig. 3. Influences of pH and temperature on the [14C]tyramine uptake in hepatocytes. The transport experiments were performed in the medium at various pH (A) at either 4°C ( • ) or 37°C ( [] ) and at different temperatures (B). The testing steps are those given in Materials and Methods.



Inhibition of [ 14C]tyramine uptake by different substances

The uptake of tyramine (6.6 #M) in hepatocytes was measured in the presence of the different substances. All the steps were performed as described in Materials and Methods. The concentration used for the compounds producing 'no effect' is 10 -3 M. It is to be noted that none of the tested compounds affects the diffusion component of the uptake. Where S.E.s are given, the point is the mean value of at least three determinations from two animals. The statistic analysis showed that there were no significant differences for the ICs0 between L-epinephrine and DL-epinephrine as well as between L-norepinephfine and DL-norepinephrine. However, the difference for ICs0 between epinephrine and norepinephrine was significant (P < 0.05). Substance Benztropine Imipramine Atropine Clomipramine Chlorpheniramine Desipramine Chlorpromazine DL-Propranolol Tryptamine Cocaine Amphetamine Phentolamine Tyramine Dopamine Phenethylamine Metaraminol 5-Hydroxytryptamine DJ.-Octopamine DL-Epinephrine L-Dopa c-Isoproterenol Indole Cimetidine Ephedrine Phenylethanolamine 5-Hydoxyindole L-Norepinephrine DL-Norepinephrine Nicotine Carbacholine Histamine Acetylcholine Choline Tyramine-cellobiose Diethylstibestrol Histidine Phenylalanine Tyrosine Tryptophan Ethylamine Reserpine

ICso (M) (X ± S.D.) 3.7.10 - 7 ± 4.9" 10 s 5.5.10 7 ± 1.5.10-7 1.0- 10 6 1.0- 10 6 1 . 2 - 1 0 - 6 ± 2.4' 10 7 3.0.10 6 5.0.10 6 8.0- 10- 6 8.0.10 - 6 1.1.10 -5 1.8.10-s 2.8.10- 5 3.1.10 s±1.3.10 -s 5.2-10 -5 ±6.5.10 6 8.4.10- 5 ± 4.5- 10 -6 1.1.10- 4 1.4.10 -4 ± 2.4.10 s 1.7.10- 4 1.7' 10 - 4 ± 1.2" 10 4 2.0" 10 - 4

3.0.10-4 + 1.2.10 - 4 3.2.10-4 4.2. ! 0 - 4 4.4.10 - 4 4.8.10 4 6.0.10 - 4 7.6.10 - 4 ±5.6.10 -s 9.0-10-4 ± 1.5.10 - 4 > 1.0.10- 3 No effect No effect No effect No effect No effect No effect No effect No effect No effect No effect No effect No effect

t h e u p t a k e o f t y r a m i n e is slightly b u t significantly inh i b i t e d , a n d is totally a b o l i s h e d by SO42- .

a m i n e s can inhibit the u p t a k e of t y r a m i n c by h e p a t o cytes, but a m i n o acids and H, N and M r e c e p t o r agonists, for e x a m p l e , tyrosine, t r y p t o p h a n , histamine, nicotine a n d c a r b a c h o l i n e , have no effect. In T a b l e IV, we show the s t r u c t u r a l r e q u i r e m e n t s for the inhibition of t y r a m i n e u p t a k e by its a n a l o g u e s taking p h e n e t h y l a m i n e as the p a r e n t structure. T h e results indicate that: (1) the i n t r o d u c t i o n of a p h e n o l i c hydroxyl g r o u p in p o s i t i o n 4 e n h a n c e s the affinity for the u p t a k e site. T h e p r e s e n c e of a s e c o n d p h e n o l i c hydroxyl g r o u p in p o s i t i o n 3 has no f u r t h e r additive effect in increasing the affinity; (2) the p r e s e n c e of a /3-hydroxyl g r o u p r e d u c e s the affinity. This effect is relatively large when two p h e n o l i c hydroxyl g r o u p s a l r e a d y exist in positions 3 a n d 4, respectively; (3) in the p r e s e n c e of an a - m e t h y l g r o u p the affinity increases. However, in all the cases, with or w i t h o u t the p r e s e n c e of p h e n o l i c hydroxyl groups, the affinities are largely d e p r e s s e d or comp l e t e l y a b o l i s h e d w h e n an a - c a r b o x y l g r o u p is introd u c e d ; (4) the effect o f N - a l k y l a t i o n increases the affinity o f that m o l e c u l e . O t h e r s t r u c t u r a l c h a n g e s can also i n f l u e n c e the affinity. If a b e n z e n e ring is r e p l a c e d by an i m i d a z o l e ring, the affinity d i s a p p e a r s , In contrast, w h e n it is r e p l a c e d by an i n d o l e ring, the affinity is sharply e n h a n c e d . F u r t h e r m o r e , if a hydroxyl g r o u p is i n t r o d u c e d in p o s i t i o n 5 o f the i n d o l e ring the affinity is lost.

Effects of amino acid modifiers T o gain insight into the m o l e c u l a r c h a r a c t e r i s t i c s of t h e t y r a m i n e t r a n s p o r t we have i n v e s t i g a t e d the sensitivity o f t h e c a r r i e r to p r o t e i n modifiers. A n u m b e r o f m o d i f i e r s have b e e n selected, b o t h on the basis o f assay c o n d i t i o n s which d o not p e r se l e a d to inactivation, as well as on t h e basis o f selectivity, c o n s i d e r i n g t h a t s o m e a m i n o acids are m o r e often e n c o u n t e r e d at t h e active site ( b i n d i n g site) o f p r o t e i n s i n t e r a c t i n g with s u b s t r a t e s [7-10]. T h e results listed in T a b l e V show t h a t significant inhibition o f t y r a m i n e t r a n s p o r t in h e p atocytes was achieved by eight types o f cysteine covalent modifiers. A m o n g them, N E M has the highest affinity for this carrier. S o m e o f the thiol g r o u p r e a c tive agents, P I T C for instance, u n d e r the c o n d i t i o n s e m p l o y e d , can also r e a c t with the lysine residues. However, the lysine-specific r e a g e n t s d o not inhibit tyram i n e t r a n s p o r t ( E A I ) or have a l i m i t e d inhibiting effect even at high c o n c e n t r a t i o n (TNBS). M o d i f i e r s for r e s i d u e s of t r y p t o p h a n (NBS) a n d a r g i n i n e ( P G O ) only have a slight inhibiting effect on the t r a n s p o r t , while D E P , a histidine r e s i d u e modifier, does not affect the uptake. Discussion

Inhibition of tyramine uptake in hepatocytes T h e results r e p o r t e d in T a b l e I I I show t h a t n u m e r ous t y r a m i n e n o n - a n a l o g u e s a n d s y m p a t h o m i m e t i c

K i n e t i c results show t h a t t y r a m i n e can e n t e r h e p a t o cytes by s i m p l e diffusion a n d by a t r a n s p o r t e r - m e d i a t e d

81 process requiring an activation energy of 1 2.9 kcal/ mol. The fact that a few min after adding tyramine to cell suspension its intracellular concentration is 17times higher than that of the medium suggests that active transport is involved. Such a hypothesis is supported by observations that cyanide, azide, oligomycin and dinitrophenol inhibit tyramine entry to some extent. The transporter-mediated uptake of tyramine by hepatocytes can be completely depressed by FCCP, a protonophore, but at a concentration that can have non-specific deleterious effects on cells. More significant is the fact that several anions NO3, SCN- and SO42- which can change the normal membrane potential, inhibit tyramine transport, suggesting that the main force driving tyramine transport is membrane potential. The effect on tyramine penetration of covalent side-chain protein modifiers brings additional evidence to the presence of a tyramine transporter in the hepatocyte plasma membrane by giving some information on its structural requirements to be efficient. NEM, at relatively low concentration, suppresses tyramine transport to a large extent, indicating that cysteine is essen-

tial for a normal carrier function. On the other hand, the null or small effect of other side-chain amino modifiers illustrate that histidine, tryptophan, arginine and lysine are not directly concerned with the binding reaction. Inhibition studies indicate that the hepatocyte tyramine carrier is definitely not involved in amino acid transport. However, they show that several amines which exhibit, like dopamine and tryptamine, or do not exhibit, like benztropine and imipramine, structural homology with tyramine are potent inhibitors of its transport at low concentration. Therefore we cannot exclude the possibility that tyramine carrier might be used by hepatocytes for the transport of various amines. This hypothesis is currently being investigated. From a pharmacological point of view, these observations suggest that some drugs, used in therapeutic medicine, could interfere with the uptake of tyramine by the liver. For example, the efficient antidepressant concentration of imipramine in plasma is between 0.3 and 1 /zM [14], and a propranolol plasma concentration of up to 4 /xM is required in some ventricular arrhythmias [15]. These concentrations are similar to that causing half inhibition of tyramine uptake in our


Structural requirements for the inhibition of tyramine uptake by hepatocytes Substance

5 4


~--~.._.f 3



Relative affinity










Amphetamine Tyramine Dopamine Phenethylamine Octopamine L-Dopa (-)-Epinephrine Isoproterenol Phenylethanolamine (-)-Norepinephrine Phenylalanine Tyrosine Tyramine-cellobiose





H H H H H H CH 3 CH(CH3) 2 H H H H












[email protected]



467.0 271.0 162.0 100.0 49.4 42.0 28.0 28.0 17.5 11.1 0.0 0.0 0.0













* They replace the benzene ring of the parent molecule.


Effects of amino acid modifiers on the uptake of 14C-tyramine hepatocytes The experimental conditions were selected on the basis of the chemical reaction requirements and the tolerance of the carrier to the reaction conditions. 3.106 hepatocytes were incubated in 2 ml perfusion buffer at 25°C with NEM (pH 7.6) or NBS (pH 6.0) for 20 min, or PGO at pH 7.6 for 60 min in a dark room; or DEP (pH 6.0) or pCMB (pH 7.0) for 30 min, or with EMTSA, pAPMA, PITC, iodoacetamide, PMC, pCMPS, EAI and TNBS, respectively, at pH 7.6 for 30 min. The cells were washed two times with 5 ml perfusion buffer at 4°C before the transport measurement was performed in the same medium. Amino acid selectivity


Concentration (M)



10 -5 5.10 -5 10 -4 10 -3 10 -5 10 -4 10 3 10 5 10 -4 10 -3 10 -4 10 3 4" 10 3 8"10 -3 10 4


2.10- 4 10 - 3

88 20 14 0 82 26 1 68 50 0 96 35 14 0 90 30 32 95 17 8 3


10 - 3 2.10 -3 10 - 3 2.10 -3

106 102 46 50





5" 10 - 4


10 -3 10 -4 10


Tyramine transport (% of control)





10 - 3 2"10 -3 4" 10 -3

82 87 69



5" 10 -5 10 4

81 86






10 - 3


10- 3 2.10 -3

109 112

experiment. Hence, intake of such drugs can inhibit tyramine uptake by the liw=r.

Acknowledgements This work was supported by the Fonds National de la Recherche Scientifique, the Fonds de la Recherche Scientifique Medicale (contract No. 3.452391) and the ASBL Air Escargot.

References 1 Johnson, R.G., Carty, S.E., Hayflick, S. and Scarpa, A. (1982) Biochem. Pharmacol. 31, 815-823. 2 Blackwell, B. (1963) Lancet ii, 849-851. 3 Hanington, E. (1967) Br. Med. J. 2, 550-558. 4 Seglen, P.O. (1976) Methods Cell Biol. 13, 29-83. 5 Burgen, A.S.V. and Iversen, L.L. (1965) Br. J. Pharmacol. 25, 34-49. 6 Clow, A., Glover, V., Oxenkrug, G.F. and Sandier, M. (1989) Neurosci. Lett. 107, 331-334. 7 Mancini, G.M.S., de Jonge, H.R., Galjarrd, H. and Verheijen, F.W. (1989) J. Biol. Chem. 264, 15247-15254. 8 Riordan, J.F., McElvany, K.D. and Borders, C.L., Jr. (1977) Science 195, 884-886. 9 Vedland, M., Waheed, A., Von Figwra, K. and Pohlmann, R. (1991) J. Biol. Chem. 266, 2917-2923. 10 Bateman, R.C. Jr., Jackson, D., Slaughter, C.A., Unnithan, S., Chai, Y. G., Moomaw, C. and Hersh, L.B. (1989) J. Biol. Chem. 264, 6151-6157. 11 Loud, A.V. (1968)J. Cell Biol. 37, 27-46. 12 Noji, S., Sato, Y., Suzuki, R. and Taniguchi, S. (1988) J. Biochem. 175, 491-495. 13 Johnson, R.G., Cartty, S.E. and Scarpa, A. (1981) J. Biol. Chem. 256, 5773-5780. 14 Glassman, A.H., Perel, J.M., Shostak, M., Kantor, S.J. and Fleiss, J.L. (1977) Arch. Gen. Psychiatry 34, 1 97-204. 15 Regardh, C.G. (1982) Acta Med. Scand. 665, Suppl., 49-60.

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