/ . Biochem., 78, 821-824 (1975)

Ascorbate 2-Sulfate Inhibits Dopamine /3-Hydroxylase Reaction, but not Ascorbate Oxidase Reaction Hiroshi HATANAKA,* Fujio EGAMI,* Takeshi KATO,** and Toshiharu NAGATSU** *Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, and ••Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Chikusa-ku, Nagoya, Aichi 464 Received for publication, May 27, 1975

Bovine adrenal dopamine /3-hydroxylase [EC 1.14.17.1] was considerably inhibited by ascorbate 2-sulfate. The inhibition was competitive with regard to ascorbate. The Ki value was 3.44 mM. The possibility that ascorbate 2-sulfate may play a regulatory role in the biosynthesis of norepinephrine is suggested. Another copper-containing oxidase, squash ascorbate oxidase [EC 1.10.3.3], was not inhibited by the same compound at a concentration of 150 mM.

Since ascorbate 2-sulfate was discovered in various animal organs (1—4), its physiological significance has been discussed by many investigators (1—7). We started biochemical studies on ascorbate 2-sulfate to investigate two hypotheses, i.e., that ascorbate 2-sulfate is the physiologically active form as a sulfating agent, or that the physiologically active form is ascorbate and ascorbate 2-sulfate regulates the pool size of ascorbate. In fact, ascorbate 2-sulfate was shown to serve as a sulfating agent under various conditions in vitro or in vivo either directly or indirectly (5—11). Moreover, ascorbate 2-sulfate, being much more stable than ascorbate (12—14), may be a storage form in vivo and serve as either an ascorbate source or as a sulfate source through enzymatic hydrolysis. Ascorbate 2-sulfate might be synthesized by sulfate transfer from 3'-phosphoadenylyl sulfate (15) and, when necessary, might be hydrolyzed by arylsulfatase Vol. 78, No. 4, 1975

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A [EC 3.1.6.1] or ascorbate-2-sulfate sulfohydrolase to give ascorbate and sulfate (12, 16-19). It is also of interest whether ascorbate 2sulfate may act not only as an ascorbate or sulfate source as mentioned above, but also as a metabolic regulator for reactions in which ascorbate participates. The present paper suggests the latter possibility in the case of the bovine adrenal dopamine j8-hydroxylase reaction. MATERIALS AND METHODS Dopamine fi-Hydroxylase and Its Assay— Dopamine /?-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate : oxygen oxidoreductase (0-hydroxylating), EC 1.14.17. l] was purified to homogeneity from the bovine adrenal medulla essentially by the method of Friedman and Kaufman. (20). Its enzyme activity was

H. HATANAKA, F. EGAMI, T. KATO, and T. NAGATSU

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assayed by photometrically measuring octopamine formed from tyramine, according to Nagatsu and Udenfriend (21). The reaction mixture, in a total volume of 1 ml, contained 20 /imoles of tyramine-HCl, 10 //moles of sodium fumarate, a suitable amount of ascorbic acid, 200 //moles of sodium acetate-acetic acid buffer, pH 5.0, 20 /*g of bovine serum albumin, 50 fig of catalase [EC 1.11.1.6], and bovine adrenal dopamine /3-hydroxylase. The reaction was carried out for 1 hr at 37°. Ascorbate Oxidase and Its Assay—Green zucchini squash ascorbate oxidase [L-ascorbate: oxygen oxidoreductase, EC 1.10. 3. 3] was kindly provided by Dr. C. R. Dawson of Columbia University. Its enzyme activity was measured by the method of Dawson and Magee (22) with some modifications. The reaction mixture, in a total volume of 3 ml, contained 20 //I of 0.1 M sodium ascorbate solution containing 50 mg of metaphosphoric acid per ml, 1.5 mg of bovine serum albumin, 2 ml of 0.2 M Na2HPO4-0.1 M citric acid buffer, pH 5.7, and an aliquot of squash ascorbate oxidase. The rate of oxygen consumption during the oxidation of ascorbate was measured by means of a Warburg-type manometer as described in the literature. In this study, we used an oxygen probe (Yellow Spring Inst, type YSI 5331). The reaction was carried out at 25° with continuous stirring. Under the above assay conditions, the enzyme concentration curve was linear up to at least 240 ng/ml. The specific activity of ascorbate oxidase used in the present investigation was estimated to be 1.01 X 103 //moles of oxygen consumed per minute per mg of protein.

1

2 I/S

3 ( 1/mM )

4

5

Fig. 1. Effect of ascorbate 2-sulfate on bovine adrenal dopamine /3-hydroxylase activity. Experimental conditions are described in the text. Control, filled circles; 3 mM of ascorbate 2-sulfate, open circles.

COMPLETE -ENZYME,-ASCORBATE, +ASCORBATE 2-SULFATE

lOOnmoles of OXYGEN

RESULTS

Fig. 2. Rate of oxygen consumption during the oxidation of ascorbate by squash ascorbate oxidase. Experimental conditions are described in the text. The amounts of enzyme etc. used in this experiment are listed under Exp. Ill in Table I.

Kinetic Representation of Ascorbate 2-Sulfate Inhibition of Bovine Adrenal Dopamine fiHydroxylase Activity—Dopamine /3-hydroxylase has been obtained in homogenous form from bovine adrenal glands and was shown to be a •copper protein (20). This enzyme is known to require ascorbic acid in order to reduce ions. Figure 1 represents the results of a kinetic study of ascorbate 2-sulfate inhibition of this enzyme activity. Lineweaver-Burk plots show-

ed that ascorbate 2-sulfate inhibited the enzyme reaction competitively with regard to ascorbate. The K{ value was 3.44 mM. No Inhibition by Ascorbate 2-Sulfate of the Squash Ascorbate Oxidase Reaction—For comparison with the inhibition of bovine adrenal dopamine ^-hydroxylase activity by ascorbate 2-sulfate, we examined the effect of ascorbate 2-sulfate on another enzyme affecting ascorbate. / . Biochem.

ASCORBATE 2-SULFATE INHIBITION OF DOPAMINE 0-HYDROXYLASE

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TABLE I. Effect of ascorbate 2-sulfate on squash ascorbate oxidase activity. Experimental conditions are described in the text. The amount of enzyme used in this experiment was 120 ng of protein. Enzyme activity / nmoles oxygen \ \consumed per min/

Exp. No.

Experimental conditions

Exp. I Exp. II

After 2 min in complete minus enzyme, enzyme was added. After 2 min in complete minus enzyme and plus 450 //moles of ascorbate 2-sulfate, enzyme was added. After 2 min in complete minus enzyme and ascorbate, but plus 90 //moles of ascorbate 2-sulfate, enzyme was added. After 2 min more under the above conditions, ascorbate was added.

Exp. Ill

As can be seen in Table I, squash ascorbate oxidase was not inhibited by ascorbate 2-sulfate at a concentration of 150 mM (Exp. I and II). Oxygen consumption during the enzyme reaction was detected by means of an oxygen probe, as shown in Fig. 2. Figure 2 shows that ascorbate oxidase was not able to oxidize ascorbate 2-sulfate instead of ascorbate. The experimental data in Fig. 2 are listed under Exp. Ill of Table I. DISCUSSION Bovine adrenal dopamine j9-hydroxylase has been established to be a mixed function oxidase ( 23). The stoichiometry of the reaction showed that an electron donor such as ascorbate is an essential cofactor for the hydroxylase and is oxidized stoichiometrically during the reaction. A large amount of ascorbate is known to exist in mammalian adrenal glands. Its physiological function is not fully clarified, but one of the functions is considered to be the role of cofactor in the dopamine ^-hydroxylase reaction. This role depends on the strong reducing power of ascorbate, which reduces cupric ions attached to the enzyme. The existence of ascorbate 2-sulfate in mammalian organs including adrenal glands suggests an antagonistic effect against ascorbate. In the present communication, we have shown that ascorbate 2-sulfate competed with ascorbate in the dopamine ^-hydroxylase reaction. This observation led us to postulate that ascorbate 2-sulfate might be a specific regulator for dopamine ^-hydroxylase in bovine adrenal glands. Vol. 78, No. 4, 1975

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A similar copper-containing enzyme, ascorbate oxidase from green zucchini squash, was not inhibited by ascorbate 2-sulfate. This result suggests that the inhibition or noninhibition by ascorbate 2-sulfate depends upon the nature of copper enzymes affecting ascorbate. Conflicting results on the antiscorbutic effect of ascorbate 2-sulfate in guinea pigs have been reported (24, 25). Ascorbate is thought to participate in collagen biosynthesis, probably as a cofactor in the protocollagen proline hydroxylase [EC 1.14.11. 2] reaction (26). From the results obtained in this communication, ascorbate 2-sulfate is expected to regulate the reaction, perhaps competing with ascorbate. Further studies are required to confirm that ascorbate 2-sulfate may regulate reactions involving ascorbate as a metabolic inhibitor. The authors wish to express their gratitude to Prof. C.R. Dawson (Columbia University) for a generous supply of squash ascorbate oxidase. The authors are also indebted to Dr. S. Nakamura (Kitasato University) and Dr. T. Oshima (Mitsubishi-Kasei Institute of Life Sciences) for helpful discussions. REFERENCES 1. Mead, C.G. & Finamore, F.J. (1969) Biochemistry 8, 2652-2655 2. Baker, E.M., Hammer, D.C., March, S.C., Tolbert, B.M., & Canham, J.E. (1971) Science 173, 826-827 3. Mumma, R.O. & Verlangieri, A.J. (1972) Biochim. Biophys. Ada 273, 249-253 4. Horning, D., Gallo-Torres, H.E., & Weiser, H. (1973) Biochim. Biophys. Ada 320, 549-556

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5. Ford, E.A. & Ruoff, P.M. (1965) Chem. Commun. 24, 630-631 6. Chu, T.M. & Slaunwhite, W.R., Jr. (1968) Steroids 12, 309-321 7. Murama, R.O. (1968) Biochim. Biophys. Ada 165, 571-573 8. Verlangieri, A J . & Mumma, R.O. (1973) Atherosclerosis 17, 37-48 9. Horning, D., Weber, F., & Wiss, O. (1974) Z. Klin. Chem. Klin. Biochem. 12, 62-65 10. Hatanaka, H., Yamagata, T., & Egami, F. (1974) Proc. Japan Acad. 50, 747-750 11. Shapiro, S.S. & Poon, J.P. (1975) Biochim. Biophys. Ada 385, 221-231 12. Roy, A.B. (1975) Biochim. Biophys. Ada 377, 356-363 13. Quadri, S.F., Seib, P.A., & Deyoe, C.W. (1973) Carbohyd. Res. 29, 259-264 14. Seib, P.A., Liang, Y.-T., Lee, C.-H., Hoseney, R.C., & Deyoe, C.W. (1974) / . Chem. Soc, Perkin I 1220-1224 15. Tolbert, B.M., Grimes, W.M., & Baker, E.M. (1974) Federation Proc. 33, 666 16. Hatanaka, H., Ogawa, Y., & Egami, F. (1974) / . Biochem. 75, 861-866

17. Hatanaka, H., Ogawa, Y., & Egami, F. (1975) / . Biochem. 77, 353-359 18. Hatanaka, H., Ogawa, Y., & Egami, F. (1975) / . Biochem. 77, 801-806 19. Hatanaka, H., Ogawa, Y., & Egami, F. (1975) / . Biochem. 77, 807-810 20. Friedman, S. & Kaufman, S. (1965)/. Biol. Chem. 240, 4763-4773 21. Nagatsu, T. & Udenfriend, S. (1972) Clin. Chem. 18, 980-983 22. Dawson, C.R. & Magee, R.J. (1955) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 2, pp. 831-835, Academic Press, New York 23. Levin, E.Y., Levenberg, B., & Kaufman, S. (1960) / . Biol. Chem. 235, 2080-2086 24. Mumma, R.O., Mckee, E.E., Verlangieri, A.J., & Barron, G.P. (1972) Nutr. Rep. Int. 6, 133-137 25. Kueniz, W., Avenia, R., & Kamm, J.J. (1974) / . Nutr. 140, 952-956 26. Barnes, M.J. & Kodicek, E. (1972) Vitamins and Hormones 30, 1-43

/ . Biochem.

Ascorbate 2-sulfate inhibits dopamine beta-hydroxylase reaction, but not ascorbate oxidase reaction.

Bovine adrenal dopamine beta-hydroxylase [EC 1.14.17.1] was considerably inhibited by ascorbate 2-sulfate. The inhibition was competitive with regard ...
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