PRELIMINARY COMMUNICATION

/ . Biochem., 78, 427-429 (1975)

Charonia lampas Arylsulfatase1 Hiroshi HATANAKA,* Yoko OGAWA,* Fujio EGAMI,* Ineo ISHIZUKA,** and Yoshitaka NAGAI** *Mitsubishi-Kasei Institute of Life Sciences, Minamiooya, Machida-shi, Tokyo 194, and **Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173 Received for publication, May 15, 1975

The activities of arylsulfatase [EC 3.1.6.1] and glycosulfatase [EC 3.1.6.3] from the liver of Charonia lampas were almost completely separated from each other. Sulfate ester bonds at position 3 of the galactose moiety of sulfatide and seminolipid were easily hydrolyzed by arylsulfatase, but scarcely affected by glycosulfatase.

Mammalian arylsulfatase A [EC 3.1.6.1] is known to show sulfatide sulfohydrolase [EC 3.1.6.8] activity (2,3). In addition, the testicular sulfoglycerogalactolipid, seminolipid, was recently reported to be hydrolyzed by the same enzyme (4, 5). This seems rather remarkable, because the sulfate group of this lipid is attached to the C-3 hydroxyl group of the galactose moiety (6, 7). On the other hand, no studies have been made on whether these sulfogalactolipids can be hydrolyzed by glycosulfatase, which is known to hydrolyze the sulfate ester linkage at position 6 of glucose (8). This is probably because the occurrence of glycosulfatase in higher animals is still doubtful (8). To examine this problem the present experiments were undertaken with enzyme preparations from the liver (hepatopancreas) of the marine gastropod,

Charonia lampas, which is known to have high glycosulfatase and arylsulfatase activities (9, 10). As shown in this paper, both sulfatide and seminolipid were easily hydrolyzed by the arylsulfatase from the liver of C. lampas, but were scarcely affected by the glycosulfatase [EC 3.1.6. 3]. Three species of sulfatases were separated from the liver of C. lampas, glycosulfatase I and II, and arylsulfatase. Details of the methods of purification and characterization of the enzymes will be reported elsewhere.2 Arylsulfatase was purified essentially as described in a previous paper {11), and was further separated from glycosulfatase by electrofocussing. Glycosulfatase I and II were separated from each other using a column of Concananavalin A-Sepharose, on which only the latter was retained.

1 This paper is No. V of the series " Ascorbate-2sulfate-Sulfohydrolase." The preceding paper in this series is Ref. /.

Vol. 78, No. 2, 1975

2

H. Hatanaka, Y. Ogawa, and F: Egami, manuscript in preparation.

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Sulfatide and Seminolipid as Substrates of

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PRELIMINARY COMMUNICATION graphy after separation by thin layer chromatography on silica gel (5). Sulfatide and seminolipid sulfohydrolase activities were measured essentially as described by Porter et al. (12). Ascorbate-2sulfate sulfohydrolase, arylsulfatase, and glycosulfatase activities were measured as described previously (11, 13). Figure 1 shows the pH-activity profiles of the three purified enzymes, glycosulfatase I and II, and arylsulfatase with />-nitrophenyl sulfate and glucose 6-sulfate as substrates. The glycosulfatase I and II preparations showed weak activities with ^-nitrophenyl

Fig. 1. pH-Activity curves of the three sulfatases from C. lampas liver with p-nitrophenyl sulfate and glucose 6-sulfate as substrates. With />-nitrophenyl sulfate (broken line) as substrate, the assay mixture in a total volume of 300 //I, contained 3 //moles of />-nitrophenyl sulfate, 30 //moles of buffer, and enzyme. With glucose 6-sulfate (solid line) as substrate, the mixture in a total volume of 200 //I, contained 5 //moles of glucose 6-sulfate, 20 //moles of buffer, and enzyme. Enzyme activities were measured in sodium acetate-acetic acid buffer (filled circles, pH 4.0-5.5), Tris-acetic acid buffer (half-filled circles, pH 6.0-7.5), and Tris-HCl buffer (open circles, pH 7.5-9.0). The enzyme preparations used were glycosulfatase I (a), glycosulfatase II (b), and arylsulfatase (c). One unit of enzyme activity represents one micromole of substrate hydrolyzed per min. TABLE I. Activities of the three sulfatases from C. lampas liver on the sulfate esters, ascorbate 2-sulfate, sulfatide, and seminolipid. For ascorbate 2-sulfate, the assay mixture in a total volume of 300 //I, contained 3 //moles of ascorbate 2-sulfate, 30 //moles of sodium acetate-acetic acid buffer, pH 4.0, 30 nmoles of 2,6dichlorindophenol, and enzyme. For sulfatide, the mixture in a total volume of 200 //I, contained 18 nmoles of rat brain [35S]sulfatide (379 dpm/nmole), 4 //moles of MnCl2, 250 //g of sodium taurodeoxycholate, 24 //moles of sodium acetate-acetic acid buffer, pH 5.0 and enzyme. For seminolipid the mixture in a- total volume of 200 //I, contained 19 nmoles of rat testicular [35S]seminolipid (341 dpm/nmole), 4 //moles of MnCl2, 250 fig of sodium taurodeoxycholate, 24 //moles of sodium acetate-acetic acid buffer, pH 5.5, and enzyme. One unit of activity represents one micromole of substrate hydrolyzed per min. Sulfate ester-hydrolyzing activities (units/mg protein) Enzyme preparation Glycosulfatase I Glycosulfatase II Arylsulfatase a

Ascorbate 2-sulfate

Sulfatide

Seminolipid

3.-51x10-'

1.15X10"4 2.61 x 10"1 1.36x10"'

1.61X10-4 1.69x10"* 2.98X10"1

Activity less than 5x10-* unit/mg protein. / . Biockem.

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[35S]Sulfatide and [35S]seminolipid were prepared from lipid extracts of the brains of 3-week old rats, 24 hr after intraperitoneal injection of [35S]sulfuric acid, as previously described ( 7 ) . Sulfogalactolipids were purified by successive column chromatographies on Florisil and DEAE-Sephadex A-25 as will be reported elsewhere, and were proved to be homogeneous by radioscanning or radioauto-

HYDROLYSIS OF SULFATIDE BY C. lampas ARYLSULFATASE

Vol. 78, No. 2, 1975

ascorbate 2-sulfate, and that glycosulfatase is inactive on such natural sugar sulfates in spite of its name. The results also show that the nomenclature of sulfatases must be revised and that further studies are required on their substrate specificities using various sugar sulfate esters. REFERENCES 1. Hatanaka, H., Ogawa, Y., & Egami, F. (1975) / . Biochem. 77, 807-810 2. Mehl, E. & Jatzkewitz, H. (1968) Biochim. Biophys. Ada 151, 619-627 3. Jerfy, A. & Roy, A.B. (1973) Biochim. Biophys. Ada 293, 178-190 4. Yamato, K., Handa, S., & Yamakawa, T. (1974) / . Biochem. 75, 1241-1247 5. Fluharty, A.L., Stevens, R.L., Miller, R.T., & Kihara, H. (1974) Biochem. Biophys. Res. Commun. 61, 348-354 6. Yamakawa, T., Kiso, N., Handa, S., Makita, A., & Yobjyama, S. (1962) / . Biochem. 52, 226-228 7. Ishizuka, I., Suzuki, M., & Yamakawa, T. (1973) / . Biochem. 73, 77-87 8. Roy, A.B. (1971) in The Enzymes (Boyer, P.D., ed.) Vol. 5, pp. 1-19, Academic Press, New York 9. Takahashi, N. & Egami, F. (1961) Biochem. J. 80, 384-386 10. Nishida-Fukuda, M. & Egami, F. (1970) Biochem. J. 119, 39-47 11. Hatanaka, H., Ogawa, Y., & Egami, F. (1975) / . Biochem. 77, 353-359 12. Porter, M.T., Fluharty, A.L., De La Flor, S.D., & Kihara, H. (1972) Biochim. Biophys. Ada 258, 769-778 13. Hatanaka, H., Ogawa, Y., & Egami, F. (1974) / . Biochem. 75, 861-866 14. Nicholls, R.G. & Roy, A.B. (1971) in The Enzymes (Boy er, P.D., ed.) Vol.5, pp.21-41, Academic Press, New York

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sulfate with optima at pH 5.0 (Fig. 1-a, b). These activities are probably not due to contamination with arylsulfatase because it has a pH optimum of 7.5, as shown in Fig. 1-c. In a separate experiment, the arylsulfatase preparation showed slight activity with glucose 6-sulfate at pH 5.5 (specific activity, 0.032 units per mg protein.) It is uncertain whether this is due to contamination with glycosulfatase or to very weak activity of arylsulfatase against glucose 6-sulfate. The arylsulfatase preparation hydrolyzed both brain sulfatide and testicular seminolipid, as shown in Table I, whereas the glycosulfatase I and II preparations scarcely hydrolyzed these sulfolipids. These results show that sulfatide and seminolipid were hydrolyzed by arylsulfatase from the liver of C. lampas, irrespective of the presence of a large amount of glycosulfatase. As in the case of mammalian arylsulfatase A (2—5), taurodeoxycholate and MnCl2 were required for the reaction. The preparation had pH-optima of 5.0 and 5.5 with sulfatide and seminolipid, respectively, and these are different somewhat from the value of pH 4.5 of mammalian arylsulfatase. It has been suggested that ascorbate 2sulfate, the sulfate ester of a sugar derivative, may be hydrolyzed by C. lampas arylsulfatase (11). This was confirmed in the present work (Table I). Artificial substrates, such as £-nitrocatechol sulfate and ^-nitrophenyl sulfate are most frequently used for in studies on arylsulfatase. So the physiological significance of arylsulfatase is uncertain (14). The present work indicates that arylsulfatase hydrolyzes various natural sulfate esters of sugar derivatives, such as sulfatide, seminolipid, and

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Sulfatide and seminolipid as substrates of Charonia lampas arylsulfatase.

PRELIMINARY COMMUNICATION / . Biochem., 78, 427-429 (1975) Charonia lampas Arylsulfatase1 Hiroshi HATANAKA,* Yoko OGAWA,* Fujio EGAMI,* Ineo ISHIZUK...
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