Studies of Sulfate Utilization by Algae
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Plant Physiol. (1976) 57, 430-436 Studies of Sulfate Utilization by Algae 15. ENZYMES OF ASSIMILATORY SULFATE REDUCTION IN EUGLENA AND THEIR CELLULAR LOCALIZATION' Received for publication June 10, 1975 and in revised form November 18, 1975 CHRISTIAN BRUNOLD2 AND JEROME A. SCHIFF3 Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02154 ABSTRACT been found in non-photosynthetic organisms such as Escherichia coli (35, 37) and yeast (20, 40) where the nucleotide sulfonyl Crude extracts of wild-type Euglena grown in the light (WTL) or in donor is PAPS.4 the dark (WTD) and a mutant lacking detectable plastid DNA (W3BUL) Sulfite reductases which reduce free sulfite to free sulfide have contain adenosine 5'-phosphosulfate (APS) sulfottansferase. Isotope also been found in many organisms (23) where they have been dilution experiments indicate that adenosine 3-phosphate 5'-phospho- suggested as participants in a pathway of sulfate reduction in- sulfate (PAPS) sulfotransferase is absent. volving free inorganic intermediates. In Chlorella, however, Thiosulfonate reductase, requiring addition of NADH or NADPH studies with mutants suggest that the bound pathway is the but not ferredoxin, and O-acetyl-L-serine sulfhydrylse, the two other physiological route of sulfate reduction in vivo with sulfite re- enzymes of the bound intermediate pathway of assimilatory sulfate ductase participating only when free sulfite is supplied exoge- reduction, are also present. Increasing levels of all three enzymes were nously. In E. coli the separation of the two pathways is less clear found in WTL, WTD, and W3BUL during logarithmic growth but the since thiosulfonate reductase and sulfite reductase activities may various activities were similar at comparable stages of growth in all three be contained in the same molecule (37), unlike Chlorella where types of cell. two separate enzymes appear to be present (11, 26). These results show that the three enzymes are not coded in the Since we wished to study the formation and cellular localiza- chloroplast DNA and are not restricted to Euglena cells having fully tion of the enzymes of assimilatory sulfate reduction, we turned developed chloroplasts. Consistent with this, they do not increase during to Euglena where organelle separations can be performed (5), light-induced chloroplast development in resting cells and are found to chloroplast development is under the experimenter's control and be enriched in the mitochondrial fraction. Further resolution of this mutants are available which lack chloroplast DNA (21, 22, 31). fraction on sucrose gradients shows that the APS sulfotransferase is In this paper we show that APSTase, TSRase, OASSase, and associated with both the microbody (glyoxysomal) and mitochondrial sulfite reductase activities are present in Euglena cells, correlate fractions while the thiosulfonate reductase and O-acetyl-L-serine sulfhy- these activities with the presence or absence of functional plas- drylase are associated only with the mitochondria. Thus the three known tids and the presence or absence of plastid DNA, and show that enzymes of the bound pathway of assimilatory sulfate reduction are the three enzymes are localized in the mitochondrial fraction. present in Eugkna mitochondrina. A brief abstract of this work has appeared previously (4). Although the activity of the entire bound pathway (APS to cysteine) is low in extracts, addition of dithlothreitol which releases free sulfite from MATERIALS AND METHODS the product of the APS sulfotransferase reaction, causes an increase in reduction activity indicating that a sulfite reductase is also present. It Euglena gracilis Klebs var. bacillaris Pringsheim and a mutant remains to be shown which reducing system is the significant one in vivo (W3BUL) lacking detectable chloroplast DNA (21) were culti- in Eugkna. vated aseptically in Hutner's (10) pH 3.5 medium with shaking at 26 C, as previously described (18). Dark-grown resting organisms were obtained, as described previously (32), using pH 6.8 resting medium (3). Conditions for normal light-induced chloroplast development have also been given before (33). Cell densities were determined with a model A pathway of assimilatory sulfate reduction containing car- A Coulter counter with an aperture of 100 ,um as before (41). rier-bound intermediates has been described for Chlorella where For the preparation of cell extracts, the cells were centrifuged it appears to be the major pathway of reduction in vivo (26). at 1500g for 5 min and were washed twice in 10 mm tris-HCl, pH This reduction pathway begins with adenosine 5'-phosphosul- 9.25, containing 10 mm EDTA. For routine work the washed fate, the sulfonyl group of which is transferred via APS sulfo- cells were suspended in 200 mm tris-HCl, pH 9.25, to a final transferase to a carrier to form Car-S-SO3- which is then re- density of 15 to 20 x 106 cells/ml. For assay of TSRase with G- duced to Car-S-S- by a ferredoxin-dependent thiosulfonate re- S-S03- and for measurements of reduction of APS to cysteine, ductase. The thiol group is then transferred via 0-acetyl-serine the washed cells were resuspended in 2.3 ml of 200 mm tris-HCl sulfhydrylase to OAS to form cysteine. A similar pathway has (pH 8), BSA was added to a final concentration of 0.1% (w/v), ' Research was supported by Grant BMS73-00987 AOI from the 4Abbreviations: PAPS: adenosine 3'-phosphate 5'-phosphosulfate; National Science Foundation. APS: adenosine 5'-phosphosulfate; APSTase: adenosine 5'-phospho- 2 Supported by Schweizerischer Nationalfonds. Present address: sulfate sulfotransferase; OASSase: O-acetyl-L-serine sulfhydrylase; Pflanzenphysiologisches Institut der Universitaet Bern, Altenbergrain TSRase: thiosulfonate reductase; WTL: Euglena wild type grown in the 21, 3013 Bern, Switzerland. light; WTD: Euglena wild type grown in the dark; W3BUL: Euglena 3 Abraham and Etta Goodman Professor of Biology. To whom reprint mutant lacking detectable chloroplast DNA; OAS: O-acetyl-L-serine; requests should be sent. G-S-SO3-: glutathione-S-SO3-; DTT: dithiothreitol. 430 Plant Physiol. Vol. 57, 1976 SULFATE REDUCTION IN EUGLENA ORGANELLES 431 and 50 ,lA of silicone defoamer (antifoam C emulsion, Dow Mitochondria were prepared as described by Buetow (5) from Corning diluted 1:50 with H2O) was also included, and the mutant W3BUL. For determination of enzyme activities, the suspension was gassed with N2 for 5 min. The resuspended cells pelleted mitochondria were resuspended in 200 Mm tris-HCI, pH cooled with ice water were broken in a Branson sonifier model S 9.25, to provide a final concentration of 1.5 to 3 mg/ml protein 75 at 2 amp with two bursts of 30 sec separated by an interval of and were sonicated as described above. The homogenate was 30 sec. The homogenate was centrifuged at 12,000g for 5 min, used without further centrifugation for the usual enzyme assays. and the supernatant fluid was used immediately for the enzyme For the separation of AP35S and PAP3S after incubation with assays. APSTase was measured as the production of sulfite-3S, Euglena extracts, the assay mixtures were inactivated in boiling assayed as acid volatile radioactivity from AP35S in the presence H20 for 1 min, 40 Mul of 1 N HCl were added to bring the pH to of DTT as previously described (14, 24). TSRase was measured 6.1, and after centrifugation, 50 ,l of the supernatant were as the production of H2S from dithionite or the formation of subjected to paper electrophoresis (12) in pH 5.8 citrate buffer cysteine-35S from G-S-35SO3- (25). In the dithionite assay, N2 on Whatman 3MM paper at 1800 v (30 v/cm) for 50 min at 4 C. was bubbled through the assay mixture during incubation. The AP35S and PAP35S were localized on the paper by UV, the areas gas stream was passed through 8.8 ml of zinc acetate solution containing the nucleotides were cut out, and the radioactivity (15) to remove continuously the H2S formed by the enzyme was measured with a Beckman liquid scintillation spectrometer, reaction, and sulfide was determined according to Johnson and The counting fluid was toluene-methanol (1:1) with 4 g/l PPO Nishita (15), using a molar extinction coefficient for the methyl- (Interex Corp.) and 50 mg/l POPOP (New England Nuclear ene blue formed of 35 x 103 (22). In the assay with G-S-35SO3-, Corp.). All the samples from each experiment were counted the reaction was stopped with 0.5 ml of 0.75 M formic acid-I M under identical quenching conditions. Proteins were determined acetic acid, pH 2 (8), containing 200 jig of cysteine. After according to Lowry et al. (17) using BSA as a standard. To removing the precipitated proteins by centrifugation 50 ,l of the determine Chl, a pinch of MgCO3 was added to cells washed by supernatant were subjected to paper electrophoresis on What- centrifugation in the tris-EDTA buffer used to prepare enzyme man 3 MM paper with formic-acetic buffer pH 2 (8) for 50 min extracts, and absolute acetone was used to extract the pigments. at 1800 v and 4 C. The cysteine was visualized by spraying with After adjustment of the extracts to 80% acetone with addition of 0.3% ninhydrin in 1-butanol-glacial acetic acid (100:3) and H20, absorbance was determined at 649 and 665 nm, and the heating at 100 C for 5 min (16). The spot was cut out, and its sum of Chl a and b was calculated (38). radioactivity was determined by scintillation counting. 3S-labeled sulfite and sulfate were purchased from the New OASSase was measured by the method of Becker et al (2). England Nuclear Corp. PAP35S was prepared by a modification Due to the high backgrounds in the cysteine assay caused by of the method of Hodson and Schiff (13).