A role for glutathione as an endogenous inhibitor of the nonenzymatic decarboxylation of DOPA

BIOCHEMICAL

MEDICINE

A Role of the

for

12, 137-142

(1975)

Glutathione

as an

Nonenzymatic

Endogenous

Decarboxylation

Inhibitor of DOPA

E. D. MACKOWIAK, T. A. HARE, AND w. H. VOGEL Department of Pharmaceutical Chemistry, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, and the Department of Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania Received

July 31. 1974

The aromatic L-amino acid (DOPA) is an important precursor in the formation of nemotransmitter substances (1) and of melanin (2). In both reactions, decarboxylation of DOPA is involved. Decarboxylation can occur enzymatically (3) or nonenzymatically (4,5). In most tissues nonenzymatic decarboxylation is inhibited by the presence of an unidentified endogenous inhibitor (6,7,8). In this study we have identified glutathione as a potential endogenous inhibitor of the nonenzymatic decarboxylation reaction. MATERIALS AND METHODS D,L DOPA-l-14C was obtained from AmershamSearle Corporation (0.05 mCi per 0.94 mmole), Arlington Heights, Ill. Radiochemical purity was determined by thin layer chromatography using n-butanol: 1 N acetic acid: absolute alcohol 35: 10: 10 (9). It was prepared so that each milliliter contained 1.11 x 10’ dpm per 1.4 pm. Glutathione was purchased from Sigma Chemical Company, St. Louis, MO.; N-ethylmaleimide was purchased from Mann Research Laboratories, New York and 2,2’ dithiopyridine from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. All other reagents were purchased from Fisher Scientific Co., King of Prussia, Pennsylvania. Rat liver, 10.5 g, was homogenized at 4°C with 0.1 M phosphate buffer, pH 7.4, in a 1:3 ratio. The crude homogenate was placed in a dialysis bag and dialyzed against 900 ml of distilled water for 18 hr with constant stirring at 4°C. The diffusate was concentrated by evaporation in vucuo and aliquots were added to incubation vessels containing DOPA and buffer. Incubations were performed at 37°C in a covered glass beaker with a center well (10). Incubation mixtures contained 0.04 ml D,L DOPA-l-14C, 0.86 ml phosphate buffer, pH 7.4, and 0.1 ml concentrated diffusate. 137 Copynght ,411 rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved

138

MACKOWIAK,

HARE

AND

VOGEL

Other reagents were dissolved in the phosphate buffer and added to the incubations as indicated. The volume of buffer was adjusted so that a final volume of 1.0 ml was maintained. The liberated 14C02 was absorbed by 0.2 ml of 5 N NaOH in the center well. Incubation was terminated after 1 hr by injecting 0.1 ml concentrated HCl into the reaction mixture through the rubber cap. An additional hour of incubation assured complete absorption of the 14C0, by the sodium hydroxide solution. An aliquot of the hydroxide solution was counted in 15 ml of Bray’s Solution (11) in a Packard Liquid Scintillation Counter (Model 3310 or 3375). Glutathione in the diffusates was measured either by amino acid analysis (12) or by reaction with 2,2’ dithiopyridine (13). Glutathione concentration in the crude homogenates was not measured. Solutions from incubations during the nonenzymatic decarboxylation of DOPA were analyzed using a Perkin Elmer 202 Recording Spectrophotometer. RESULTS LePlante and Tran (18) suggested that the nonenzymatic decarboxylation of DOPA is inhibited by compounds containing sulfhydryl groups. Table 1A shows the inhibition produced by various concentrations of glutathione, a sulfhydryl compound present in tissue homogenates, on the nonenzymatic decarboxylation reaction. Table 1B shows the effect of N-ethylmaleimide (NEM), a reagent that reacts with sulfhydryl groups, on the nonenzymatic reaction. NEM produced no effect on the decarboxylation itself, but did produce reversal of glutathione inhibition. Table 2 shows the effect on the reaction of the addition of 0.1 ml aliquots from diffusates of rat liver homogenates, which were concentrated loo-fold. The greatly reduced ‘“CO, production indicates the presence of a dialyzable inhibitor of the nonenzymatic reaction. Addition of 4 x 1w3 M NEM to incubation vessels containing aliquots of the diffusate partially reversed the inhibition caused by the diffusate present. The concentration of glutathione present in the concentrate of the diffusate was found to be 1.3 x 10m5M, a concentration known to be inhibitory. When the diffusate was diluted IO-fold and lOO-fold, inhibition of decarboxylation was decreased to 32% and 19% of the control value, respectively. Thus, glutathione appears to be an inhibitor of the nonenzymatic decarboxylation of DOPA. When 0.1 ml aliquots of diffusates from dialysis of the phosphate buffer only were incubated with DOPA, decarboxylation increased from 8.4 nm of 14COZ to 44.8 nm. This fivefold increase in decarboxylation was probably due to the concentration of metal ions in the phosphate buffer since the addition of 1 x lop3 M EDTA nearly abolished 14C02 production (I .4 nm 14C0,).

GLUTATHIONE

AS AN ENDOGENOUS TABLE

1

OF GLUTATHIONE (GSH) AND TV-ETHYLMALEIMIDE NONENZYMATIC DECARBOXYLATION OF

EFFECT

139

INHIBITOR

(NEM)

ON THE

DOPA

GSH

(a) Compound added

Concentration

% inhibition of YO, released

lo-3 lo-* X lo+ X lo-’

0 99 82 16 5

None (control) GSH GSH GSH GSH

1 1 1 1

X X

M M M M

GSH (1 x 10m3M) and various concentrations of NEM

(b)

Concentration of NEM

GSH GSH GSH GSH

None and NEM and NEM and NEM and NEM

% inhibition

0.8 2.0 4.0 8.0

X X X X

It3 It3 1o-3 lo’+

of “‘CO, released 0 98 63 51 50

M M M M

Incubations of 0.5 x low4 M DOPA were performed in 0.1 M phosphate buffer, pH 7.4 at 60 min and contained either glutathione and/or NEM in the concentration indicated. The addition of NEM alone in concentrations up to 8.0 x 103 M produced no significant effect on the nonenzymatic decarboxylation of DOPA. 37°C for

The absorption of DOPA in the presence of glutathione was monitored during the incubation by UV spectrophotometry. Figure 1A shows that incubation of glutathione alone using nonenzymatic conditions had an absorption peak at 210 nm which varied little during 200 minutes of incubation. Figure 1B shows that DOPA’s absorption peak at 280 nm inTABLE EFFECT

2

OF DIFFUSATES WITH AND WITHOUT NEM FROM DIALYSIS PREPARATIONS THE NONENZYMATIC DECARBOXYLATION OF DOPA

Condition

Nanomoles +ZO, per hour

Control Diffusate only” Diffusate with 4 x 10e3 M NEM

8.7 2 1.8 1.22 f 0.9 2.4 k 0.6

ON

Incubation vessels contained 0.5 x 10e4 M DOPA and 0.1 ml of diffusate from dialysis of rat liver homogenate (lOO-fold concn) in 0.1 M phosphate buffer, pH 7.4 at 37°C. Values represent the mean of 6 experiments f standard deviation. The value reported for the diffusate

with

4 x 1(r3

M NEM

is significantly

different

from

values

obtained

for

the diffusate only, p < 0.001. a Concentration

1.3 X lo-5 M.

of glutathione

present

in incubation

vessel

from

the

diffusate

was

140

MACKOWIAK,

HARE

(c, DOPA

,.,a,

AND

VOGEL

GLUTATHIONE

FIG. 1. Spectra of glutathione, DOPA, and DOPA with glutathione incubated at various times. Aliquots from three different mixtures, which were incubated at 37”C, were measured spectrophotometricahy at 0, 120, and 200 min. These incubation mixtures contained: (a) 1 x 1O-4 M glutathione in 0.1 M phosphate buffer, pH 7.4; (b) 1.2 x 10e4 M DOPA in 0.1 M phosphate buffer, pH 7.4; and- (c) 1.2 x lo+ M DOPA and 1.2 x 1O-4 M glutathione in 0.1 M phosphate buffer, pH 7.4.

tensified with increasing incubation time, but a generalized absorption which occurred throughout the spectrum caused a loss of distinction of this peak. The spectrum of-DOPA and glutathione together (Fig. 1C) shows little change in the absorption of DOPA at 280 nm and the masking of the glutathione peak at 210 nm. Increased absorption in the spectrum at wavelengths greater than 300 nm was decreased for the same incubation period, indicating retardation of the nonenzymatic reaction. No new absorption peaks formed during incubation of DOPA and glutathione together. DISCUSSION

Studies of the enzymatic and nonenzymatic decarboxylation of DOPA in rat and human tissue homogenates showed that the enzymatic (6), but not the nonenzymatic decarboxylation lead to dopamine formation (14).

GLUTATHIONE

AS

AN

ENDOGENOUS

INHIBITOR

141

An endogenous inhibitor of the nonenzymatic reaction is present both in rat (7) and human tissues which is heat stable and dialyzable (6). Many investigators studying melanin biosynthesis have postulated the presence of natural inhibitors in normal mammalian skin, human plasma, normal mouse liver, mouse tumors, and mouse melanoma. The characteristics attributed to the inhibitor(s) vary widely. Flawn and Wilde (15) have reviewed and summarized these observations and classified the inhibitor(s) into two possible categories. One group of investigators reports that the inhibitor is heat stable, dialyzable and a nonprotein sulfhydryl compound(s); others report that the inhibitor is nondialyzable, nonsulfhydryl, and protein in nature. Our experiments indicate that at least one inhibitor present in rat and human homogenates belongs to the first category. This inhibitor can be dialyzed from fresh or boiled homogenates. Diffusates which were concentrated IOO-fold by evaporation in vacua when added to incubation vessels during nonenzymatic incubations of DOPA caused inhibition of 14C0, release. Determination of glutathione concentration in the vessels either by amino acid analysis or as nonprotein sulfhydryl resulted in concentrations which are inhibitory to the nonenzymatic decarboxylation of DOPA. Furthermore, NEM, a sulfbydryl reagent, partially reversed this inhibition. Two possible mechanisms for the effectiveness of glutathione’s inhibition are: (1) The thiol may react with the double bond system either in DOPA quinone or dopachrome which occur as intermediates in the conversion of DOPA to melanin, either enzymatically or nonenzymatically (16). Kodja and Bouchilloux (17), Roston (18), and Seiji et al., (19) reported formation of a product from the reaction of the sulfhydryl group in glutathione with an oxidation product of DOPA, dopaquinone. during the enzymatic production of melanin. Although neither Seiji et al. nor we could detect such a product during nonenzymatic incubations of DOPA with glutathione, this reaction could still occur in vivo. (2) Gfutathione may be oxidized or complexed by metal ions (20,21). Addition of 1.5 x 10e4 M cupric chloride to a vessel during nonenzymatic decarboxylation of DOPA stimulated 14COZ release fivefold (7.3 + 0.2 to 39.5 5 2.9 nm 14C02). Addition of 1 x IV M glutathione to a vessel containing DOPA and copper nearly abolished all decarboxylation (0.7 t 0.3 nm 14C0,). Thus, glutathione in vitro may act by complexing metal ions, but in vivo thioether formation may also be of importance. Therefore, glutathione is an endogenous inhibitor of the nonenzymatic decarboxylation of DOPA. Since the inhibition produced by the diffusates was greater than that expected from the concentration of glutathione measured in the diffusates and since NEM did not reverse the inhibition to the degree expected, the presence of inhibitors other than glutathione is probable.

142

MACKOWIAK,

HARE

AND

VOGEL

Identification of glutathione as an endogenous inhibitor might explain the suppression of the nonenzymatic reaction that occurs in tissues but not in plasma or spinal fluid (14). Unlike tissue, plasma and spinal fluid contain little or no glutathione (22). This nonenzymatic decarboxylation could decrease the amount of L-DOPA available for brain uptake in therapy of Parkinsonism and thus represent a mode of loss of this drug in addition to its peripheral enzymatic decarboxylation. REFERENCES 1. 2. 3. 4. 5. 6.

Udenfriend, S. and Wyngaarden, J. B., Biochim. et Biophys. Acta Raper, H. S., Physiol. Rev. 8, 245 (1928). Holtz, P., Heise, R., and Ludtke, K., Arch. Exp. Path. Pharmakol. Awapara, J., Perry, T. L., Hanly, C., and Peck, E., Clin. Chim. Acta Vogel, W. H., Nuturwissenchaften, 56, 462 (1969). Vogel, W. H., Snyder. R., and Hare, T. A., Proc. Sot. Exp. Biol.

20, 48 (1956). 191, 87 (1938). 10, 286 (1964).

Med. 134, 477

(1970).

7. Rivera-Calimlin, L., Morgan, J. P., Dujovne, C. A., Bianchine, J. R., and Lasagna, L., Biochem., Pharmacol. 20,305l (1971). 8. Laplante, M., and Tran, N., Arch. Int. Pharmacodyn. 203, 249 (1973). 9. Johnson, G. A., and Boukma, S. J., Anal. Biochem. 18, 143 (1967). 10. Robins, E., Robins, J. M., Croninger, A. B., Moses, S. G., Spencer, S. J., and Hudgens, R. W., Biochem. Med. 1, 240 (1967). 11. Bray, G. W., Anal. Biochem. 1, 279 (1960). 12. Long, C. L., and Geiger, J. W., Anal. Biochem. 29, 265 (1969). 13. Grassetti, D. R., and Murray, Jr., J. F., Arch. Biochem, Biophys. 119, 41 (1967). 14. Mackowiak, E. D., Hare, T. A., and Vogel, W. H., Biochem Med. 6, 562 (1972). 15. Flawn, P. C., and Wilde, P. F., J. Invest. Derm. 55, 153 (1970). 16. Mason, H. S., J. Biol. Chem. 172, 83 (1948). 17. Kodja, A., and Bouchilloux, S., Compt. Rend. Sot. Biol. 154, 737 (1960). 18. Roston, S., J. Biol. Chem. 235, 1002 (1960). 19. Seiji, M., Yohida, T., Itakura, H., and Irimajiri, T., .I. Invest, Derm. 52, 280 (1969). 20. Voegtlin, C., Johnson, J. M., and Rosenthal, S. M., J. Biof. Chem. 93, 435 (1931). 21. Hirsch, H. M., Cancer Res. 15, 249 (1955). 22. Jocelyn, P. C., in “Biochemistry of the SH Group,” p. 10. Academic Press, New York, 1972.