Phytochemistry, Vol. 23, No. 2, pp. 251-256, 1984. 0031-9422/84 %3.00+0.00 Printed in Great Britain. 0 1984 Pergamon Press Ltd. zyxwvuts GLUTATHIONE S-TRANSFERASE FROM HE VEA BRASILIENSIS SHANMUGAM BALABASKARAN* and NARASIMAN MUNIANDY Biochemistry Department, Medical Faculty, University of Malaya, Pantai Valley, Kuala Lumpur, Malaysia (Revised received 8 July 1983) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Key Word Index-Hevea brasiIiensis; Euphorbiaceae; GSH S-transferase; latex. Abstract-Glutathione (GSH) S-transferase can be detected in a variety of tissues of Hevea brasiliensis. Lyophilized powders prepared from 20 OBO g supernatants of latex adjusted to pH 5.0 contain substantial amounts of GSH S- transferase activity which is stable at - 20” for up to 6 months. The enzyme has a broad pH optimum between 8.5 and 9.5. The K, values for GSH and l-chloro-2,4_dinitrobenzene are in the range of 3345 and 150-200 PM, respectively. The enzyme has a MW in the range of 47 000-50 OOOandan isoelectic point of 4.3. Although it appears homogeneous on analytical polyacylamide disc gel electrophoresis (PAGE) and isoelectric focusing, it resolves into five forms on DEAE- Sephadex chromatography. INTRODUCTION Glutathione S-transferase (EC 2.5.1.18) are a group of detoxicating enzymes that catalyse the conjugation of reduced glutathione (GSH) with a variety of electrophilic compounds [l--S]. These enzymes are known to be dimeric with MWs between 40000and 50000 [5]. Several groups have suggested that the GSH S-transferases consist of various pairs of three subunits designated as Ya, Yb and Yc with MWs of 22000, 23 500 and 25 000, respectively [6-91. In addition, other workers have suggested that the minimum number of subunits needed to explain the multiple forms of rat liver isoenzymes is four [lo, 111. Although the properties and distribution of GSH S- transferases have been extensively studied in vertebrates [12-161 and to some extent in invertebrates [17-201, few such studies have been conducted in plants [21-231. This may be due partly to difficulties encountered in extracting these enzymes from plant tissues. In the rubber tree Heuea brasiliensis, the cell content of the latex vessels can be easily drained by paring a portion of the bark from the tree. This white milky liquid known as latex contains, amongst other substances [24], signifi- cant amounts of GSH and other thiols [25]. Since the role of GSH in detoxication in biological systems is well known, we were led to look for glutathione-conjugating activity in latex, as well as in a variety of other tissues of the rubber tree. This paper describes the detection and distribution of GSH S-transferase in a variety of tissues from H. brasiliensis in addition to latex. The enzyme, unlike other GSH S-transferases in animals and plants, behaves unusually in that it appears as a homogeneous protein in isoelectric focusing experiments and resolves into five chromatographically distinguishable forms on DEAE anion-exchange chromatography. It is only cap- able of conjugating GSH with aryl substrates such as l- chloro-2,4_dinitrobenzene (DNCB) and 1,2-dichloro-4- nitrobenzene (DCNB). *To whom corresondence should be addressed. RESULTS Preliminary studies conducted using several prepar- ations of lyophilized powder revealed that the enzyme had a broad optimum pH between 8.5 and 9.5, with a gentle fall in activity on the acid and alkaline sides of the optimum, so that at pH values of 7.5 and 10.5 the activity was half that at the maximum. Routine enzyme assays of column eluants were conducted .in 0.1 M arginine- hydrochloric acid buffer of pH 8.3 at 28”. The enzyme was stable at 5” between pH values of 4.3-5.5 and 5.5-10.0 in 0.1 M sodium acetate-acetic acid and 0.1 M argi- nine-hydrochloric acid buffers, respectively, for up to 3 hr, which was the longest period tested. A dialysed preparation of an ammonium sulphate-fractionated enzyme lost only 20 y0 of its activity when left to stand for 12 days at 5” at pH 6.5. Even when the enzyme was exposed to 37” for 4 hr at pH 8.3 in 0.1 M arginine-hydro- chloric acid buffer it did not lose more than 10% of its activity. The specific activity of 37 different preparations of lyophilized powder prepared under similar conditions from latex collected from the same trees was 2.82 + 1.50 nkat/mg protein at 28” in the same buffer. Using different preparations of the lyophilized powder the K, for GSH was in the range 3345 PM, while that for DNCB was in the range 15&2OOpM at pH 8.3 under the same conditions. The ratio of activity for the same amount of enzyme towards DNCB and DCNB was approximately 4: 1 at pH 8.3. Most of the enzyme activity could be precipitated from aqueous solutions of the lyophilized powder between 50 and 80% saturation by ammonium sulphate resulting in a 2- to 3-fold purification. The enzyme when passed through a calibrated Sephadex G-200 column appeared as a single peak with an average MW of 47 000 + 3000. This value was obtained not only for a solution of the lyophilized powder, but also for partially purified preparations of the enzyme obtained after ammonium sulphate fractionation or anion- exchange chromatography. The MW of the enzyme was not altered when it was chromatographed on a similar column equilibrated with buffer containing either 0.1 or 0.3 M sodium chloride. 251