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Plant Physiol. (1985) 77, 296-299 0032-0889/85/77/0296/04$0 1.00/0 Characterization of Peroxisomes from the Alga Bumilleriopsis fiiformis1 Received for publication August 17, 1984 and in revised form October 17, 1984 WOLFGANG GROSS, UWE WINKLER, AND HELMUT STABENAU* Universitat Oldenburg, Fachbereich Biologie, Postfach 2503, D-2900 Oldenburg, West Germany ABSTRACT The Xa"thophycean alp Bumilkropsis filiformis possesses peroxisomes which on electron micrographs show a mostly spherical or ovoid shape with a diameter in the range of 03 micrometer. Their granular matrix is usually of moderate electron density and in a very few cases contains amorphous inclusions. No associations with other organelles could be observed. During separation in a sucrose gradient, the peroxisomes from Bumilkeiopsis equilibrate at a density of 1.22 grams per cubic centimeter. Glycolate oxidase and glyoxylate-glutamate aminotransferase were found in the isolated organelles along with catalase and uricase. However, no further leaf peroxisomal enzymes were detected. This is the first time that an ala of the group of Xaathophyceae has been demonstrated to possess a glycolate oxidase. The organelles from Bumileriopsis differ from leaf peroxisomes also by the absence of enzymes of the a-oxidation pathway. All enzymes for the degradation of fatty acids which were tested are located solely in the mitochondria. Microbodies have been demonstrated by electron microscopy to be present in many different algae (18), but only from a few of those organisms could the microbodies be isolated for biochemical characterization. Nevertheless, it has already become evident that there are different types of algal microbodies (7, 23). In Spirogyra and Mougeotia the peroxisomes are of the leaf type and are therefore involved in glycolate metabolism (22, 25, 30). On the other hand, the peroxisomes of the unicellular algae Chlorogonium, Chlamydomonas, ,Eremosphaera, and Polytomella contain neither enzymes of the glycolate metabolism nor enzymes of the glyoxylic acid cycle (6, 20, 21, 24, 27). Thus, they have been regarded as unspecialized organelles. Until now algal microbodies of the glyoxysomal type have been isolated only from heterotrophically grown Euglena (1 1). Under autotrophic conditions, the corresponding organelles of this alga like leaf peroxisomes contain enzymes of glycolate metabolism (5). But these microbodies differ from leaf peroxisomes in that they contain a glycolate dehydrogenase rather than a glycolate oxidase (4, 16). Furthermore, there are also contrary reports whether catalase is a constituent of the organelles in Euglena (3, 14, 29). Recently it was reported that the unspecialized peroxisomes from Eremosphaera and the leaftype organelles from Mougeotia both contain enzymes of the #-oxidation pathway (28). Considering also the data for higher plants (8, 9), it appeared that the possession of enzymes for the a-oxidation is a characteristic of ' Supported by the Deutsche Forschungsgemeinschaft. all different types of microbodies. This assumption now seems to be questionable since we have isolated peroxisomes from the Xanthophycean alga Bumilleriopsis which could not be demonstrated to contain enzymes for degradation of fatty acids. MATERIALS AND METHODS Algal Material and Growth Conditions. Bumilleriopsisfiliformis (Xanthophyceae), strain 802-2, was obtained from the algae collection of the Institute for Plant Physiology, University of Gottingen, W. Germany. Cultures were grown autotrophically in glass tubes at 23YC in continuous light of either 3,000 or 20,000 lux in nutrient medium (12). The cultures were aerated with air plus 2% CO2 or air only. Preparation of Cell Homogenates and Separation of Organelles. Cells of 1 L of suspension were collected by low speed centrifugation and washed with grinding medium (28). For homogenization, the cells were transferred to 20 ml of the same medium containing 100 mg Polyclar AT, 20 mg cystein, 15 mg DTT, 70 mg BSA, and 15% (w/w) sucrose. Cells were broken in a Virtis homogenizer after adding 30 ml glass beads; the homogenate was centrifuged at 5OOg for 10 min. The resulting supernatant was used for sucrose density centrifugation (28). Assays. All assays for measuring the activities ofenzymes were carried out at 25°C in 1 ml total volume. Spectrophotometric measurements were made on a Uvikon 810 photometer. Glycolate oxidase activity was measured by the reaction of glyoxylate with phenylhydrazine and by 02 consumption assayed with a Rank electrode (26). The H202 formed by the glycolate oxidase was measured by the peroxidase-aminoantipyrine reaction (13). The activity of catalase in the test medium was inhibited by addition of 0.1 mM NaN3. All other enzymes were measured as already described: catalase, Cyt c oxidase, hydroxypyruvate reductase, and malate dehydrogenase (21); uricase (24); acyl-CoA dehydrogenase, enoyl-CoA hydratase, and hydroxyacyl-CoA dehydrogenase (28); acyl-CoA oxidase (8); glutamateglyoxylate aminotransferase and serinq-glyoxylate aminotransferase (30). The amino acids formed during the aminotransferase reactions were determined by HPLC analysis (10). The protein was measured by the Folin reagent method (15). The content of Chl was measured as described by B6ger (2). Electron Microscopy. Whole cells were fixed with 4% glutaraldehyde in K-phosphate, 0.05 M (pH 8) for 1.5 h at room temperature. Then the cells were collected by low speed centrifugation and fixed for 1 h at room temperature in 2% osmium tetraoxide. Pooled peroxisomal fractions from sucrose gradients were fixed with 4% glutaraldehyde (flnal concentration) for 1 h at 4°C (1). The glutaraldehyde solution used was prepared in 45% (w/w) sucrose. Then the suspension was diluted with an equal volume of 40% (w/w) sucrose in gradient buffer and the organelles were pelleted by centrifugation for 45 min at 40,000g. After washing with the same medium the pellet was fixed for 1 h in 2.0% Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1985 American Society of 296 Plant Biologists. All rights reserved. 297 MICROBODIES FROM BUMILLERIOPSIS osmium tetraoxide in K-phosphate, 0.05 M (pH 8) containing 40% (w/w) sucrose. Fixed organelles or whole cells were dehydrated in a graded ethanol-water series and in 100% acetone. The material was embedded using a three-step infiltration in low viscosity resin (19). Sections were cut and stained with lead citrate (17). The sections then were examined in a ZEISS EM 109 electron microscope. Chemicals. All chemicals for electron microscopy and the Polyclar AT were obtained from Serva (Heidelberg, W. Germany). Glass beads (diameter: 0.25-0.30 mm) were purchased from Braun (Melsungen, W. Germany). All other chemicals were provided by Sigma (St. Louis). - c 0 0. E io RESULTS Organelles from the -crude homogenate of autotrophically grown cells were separated in a linear sucrose gradient. As indicated by the distribution of Chl and Cyt oxidase, chloroplasts and mitochondria were clearly separated equilibrating at densities of 1.18 and 1.20 g. cm-3, respectively (Fig. la, b). Since the fraction with the density of 1.20 g- cm-3 contains a relatively high activity of malate dehydrogenase, most of the mitochondria are believed to be unbroken. Microbodies in the gradient were identified by electron microscopy as well as by the distribution of catalase and uricase. Both enzymes show a main peak at a density of 1.22 g cm3 (Fig. lc). The smaller peak at a density of 1.16 g- cm-3 may possibly be due to some microbodies trapped in the chloroplast fraction. About 70% of the total activities of catalase and uricase were found in the particulate fraction. Therefore, most of the microbodies were not disrupted during homogenization and gradient centrifugation. No peak of protein was seen in the microbody fraction (density 1.22 g cm-3, Fig. la), indicating that the numUrote oxidase 30 ber of microbodies is relatively low. The microbodies from Bumilleriopsis contain a glycolate oxidizing enzyme capable of transferring electrons directly to oxygen. As shown in Table I, during this reaction, the oxygen is N reduced to H202 as in higher plants. Similar to the corresponding C w oxidase from Mougeotia (26), the enzyme shows some activity with L-lactate but not with 1-lactate as the substrates (Table I). 10 Using glycolate, a 20-fold higher activity was measured compared with L-lactate, and under this condition the Km, value determined by a Lineweaver-Burk plot was found to be 250 gM. In addition to glycolate oxidase, the microbodies from Buma Hydnocyl-CoA DH illeriopsis contain a glyoxylate-glutamate aminotransferase (Fig. o Enoyl.CoA hydr. lc). However, no aminotransferase capable of converting seine o Acyl.CoA DH(d to hydroxypyruvate was found in these organelles. For the latter X.10I Desty(203 2012 test, either glyoxylate, a-ketoglutarate, or pyruvate were used as the amino acceptors. No activity of hydroxypyruvate reductase was detected at a density of 1.22 g- cm-3 or in any other fraction of the gradient, using either NADH or NADPH. To demonstrate the presence of the fl-oxidation pathway in Bumilleriopsis the enzymes enoyl-CoA hydratase, hydroxyacylCoA dehydrogenase, acyl-CoA dehydrogenase, and acyl-CoA oxidase were assayed. No activity of acyl-CoA oxidase could be found in the fractions of the gradient measuring either the palmitoyl-CoA dependent 02 consumption or H202 production. The activity of the acyl-CoA dehydrogenase was relatively low Density (glcm3) 1.20 1.22 as compared to the other two enzymes. FIG. 1. Separation of organelles from Bumilleriopsis in a linear suAll three enzymes of the fl-oxidation demonstrated to be crose gradient. Units of enzymes per ml fraction per min: catalase, Mmol present in Bumilleriopsis were found to be constituents of the substrate; urate oxidase, nmol substrate (ordinate values [ord.v.] x 0.07 mitochondria but not of the microbodies from Bumilleriopsis = actual values [act. v.j); glutamate-glyoxylate aminotransferase, nmol (Fig. ld). substrate (ord.v. x 2.5 = act.v.); glycolate oxidase, nmol substrate (ord.v. x 0.45 = act.v.); malate dehydrogenase, 0.1 mol substrate; Cyt oxidase, DISCUSSION nmol substrate (ord.v. x 5 = act.v.); enoyl-CoA hydratase, 0.01 grmol; The microbodies from Bumilleriopsis are spheroid or ovoid in hydroxyacyl-CoA dehydrogenase, nmol substrate (ord.v. x 7.5 = act.v.); shape in most cases and their average diameter was determined acyl-CoA dehydrogenase, nmol substrate. d Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1985 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 77, 1985 GROSS ET AL. 298 Table I. Activity of Glycolate Oxidasefrom Bumilleriopsis The tests were performed with 50 mm lactate or glycolate. The enzyme was partially purified by precipitation with (NH4kSO4 (50-55% of satu- ration). D-Lactate L-Lactate Product Formed or 02 Consumed nmol/mg protein. min 0 Pyruvate 6.3 Pyruvate Glycolate Glycolate Glycolate 114.2 Glyoxylate 109.5 H202 106.5 02 Substrate A, Fijito be 0.3 ;tm (Fig. 2a). They sometimes appear somewhat elongated. The granular matrix has a higher density than that of the surrounding cytoplasm and contains amorphous inclusions in only a very few cases (Fig. 2d). No associations with other organelles could be observed as in other algae (1 8). When centrifuged to equilibrium in a sucrose density gradient, the microbodies from Bumilleriopsis were recovered at a density of 1.22 g- cm-3. On electron micrographs of the fraction 1.22 most of them appeared undamaged (Fig. 2, b-d). As demonb strated in Figure lc, the isolated organelles contain catalase and two oxidases forming the substrate for the catalase reaction. The glycolate oxidase is similar to the enzyme found in the microbodies from the Chlorophycean alga Mougeotia in that it is able to oxidize L-lactate to a small extent. The K,,, value (250 i iM) is somewhat lower than that of the enzyme from Mougeotia (415 jiM) (26) and almost the same as for the enzyme in leaf peroxisomes of higher plants (7). Uricase, the other oxidase in the peroxisomes from Bumilleriopsis, has been demonstrated to be present also in the microbodies of some unicellular algae (6, 7, 24), but not in the organelles from Mougeotia. Beside glycolate oxidase, the peroxisomes from Bumilleriopsis contain a glutamate-glyoxylate aminotransferase, and in that respect they are similar to leaf peroxisomes. However, no other leaf peroxisomal enzymes could be detected. No enzyme activity was found for formation of hydroxypyruvate from serine using different a-keto acids as the amino acceptors. In agreement with this, no activity of hydroxypyruvate reductase could be measured with either NADH or NADPH as the coenzyme. From these results it may be concluded that the physiological role of the peroxisomes in Bumilleriopsis is not the same as that of leaf s, ukilia d FIG. 2. Electron micrographs of microbodies from Bumilleriopsis. a, in situ (x 35,000); b and c, isolated organelles in the fraction with a density of 1.22 g-cm-3 (x 35,000); d, isolated organelles, one with an amorphous inclusion (x 70,000). Chl, chloroplast; M, mitochondrion; Mb, microbody; N, nucleus. peroxisomes. The peroxisomes from Bumilleriopsis are different from the leaf type organelles of Mougeotia not only by the absence of hydroxypyruvate reductase and a serine transforming aminotransferase but also by the absence of enzymes for degradation of fatty acids. In Mougeotia and Eremosphaera, three enzymes of the ,8-oxidation were found to be constituents of the microbodies and partially of the mitochondria, too (28). However, in Bumilleriopsis these enzymes are located only in the mitochondria. Therefore, in contrast to our former assumption (28), we have to conclude that the capability for degradation of fatty acids is not a common feature of all types of microbodies. LITERATURE CITED 1. BIEGLMAYER C, J GRAF, H RuIs 1973 Membranes ofglyoxysomes from castorbean endosperm. Eur J Biochem 37: 553-562 2. BOGER P 1964 Das Strukturproteid aus Chloroplasten einzelliger Grunalgen und seine Beziehung zum Chlorophyll. Flora 154: 174-21 1 3. BROWN RH, N COLLINS, MJ MERRETT 1975 Peroxidative activity in Euglena gracilis. Plant Physiol 55: 1123-1124 4. CODD GA, JM LORD, MJ MERRETT 1969 The glycolate oxidizing enzyme from algae. FEBS LeUtt 5: 341-342 5. COLLINS N, MJ MERRETT 1975 The localization of glycolate-pathway enzymes in Euglena. Biochem J 148: 321-328 6. GERHARDT B 1971 Zur Lokalisation von Enzymen der Microbodies in Poly- Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1985 American Society of Plant Biologists. All rights reserved. MICROBODIES FROM BUMILLERIOPSIS tomella caeca. Arch Mikrobiol 80: 205-218 7. GERHARDT B 1978 Microbodies/Peroxisomen pflanzlicher Zellen. SpringerVerlag, Wien, pp 78, 152-166 8. GERHARDT B 1981 Enzyme activities of the #-oxidation pathway in spinach leaf peroxisomes. FEBS Lett 126: 71-73 9. GERHARDT B 1983 Localization of ,-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues. Planta 159: 238-246 10. GODEL H, T GRASER, P F6LDI, P PFAENDER, P FURST 1984 Measurement of free amino acids in human biological fluids by HPLC. J Chromatogr 297: 49-61 1 1. Graves LB Jr, WM Becker 1974 Beta-oxidation in glyoxysomes from Euglena. J Protozool 21: 771-774 12. HESSE M 1974 Wachstum und Synchronisierung der Alge Bumilleriopsis filiformis (Xanthophyceae). Planta 120: 135-146 13. HRYB DJ, FJ HOGG 1979 Chain length specifications of peroxisomal and mitochondrial a-oxidation in rat liver. Biochem Biophys Res Commun 87: 1200-1206 14. LORD JM, MJ MERRETT 1971 The intracellular location of glycolate oxidoreductase in Euglena gracilis. Biochem J 124: 275-281 15. LOWRY OH, AL ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275 16. NELSON EB, NE TOLBERT 1970 Glycolate dehydrogenase in green algae. Arch Biochem Biophys 141: 102-1 10 17. REYNOLDS ES 1963 The use of lead citrate at high pH as an electrone-opaque stain in electron microscopy. J Cell Biol 17: 208-212 18. SILVERBERG BA 1975 An ultrastructural and cytochemical characterization of 299 microbodies in green algae. Protoplasma 83: 269-295 19. SPURR A 1969 A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 31-43 20. STABENAU H 1974a Verteilung von Microbody-Enzymen aus Chlamydomonas in Dichtegradienten. Planta 118: 35-42 21. STABENAU H 1974b Localization of enzymes of glycolate metabolism in the alga Chlorogonium elongatum. Plant Physiol 54: 921-924 22. STABENAU H 1976 Microbodies from Spirogyra. Plant Physiol 58: 693-695 23. STABENAU H 1984 Microbodies from different algae. In W Wiessner, D Robinson, PC Starr, eds, Compartments in Algal Cells and Their Interaction. Springer-Verlag, Berlin, pp 183-190 24. STABENAU H, H BEEVERS 1974 Isolation and characterization of microbodies from the alga Chlorogonium elongatum. Plant Physiol 53: 866-869 25. STABENAU H, W SAFrEL 1981 Localization of enzymes of the glycolate metabolism in Mougeotia. Ber Dtsch Bot Ges 94: 59-64 26. STABENAU H, W SAFrEL 1982 A peroxisomal glycolate oxidase in the alga Mougeotia. Planta 154: 165-167 27. STABENAU H, U WINKLER, W SAFrEL 1984a Mitochondrial metabolism of glycolate in the alga Eremosphaera viridis. Z Pflanzenphysiol 118: 413-420 28. STABENAU H, U WINKLER, W SAFTEL 1984 Enzymes of -oxidation in different types of algal microbodies. Plant Physiol 75: 531-533 29. WHITE JE, M BRODY 1974 Enzymatic characterization of sucrose-gradient microbodies of dark-grown, greening and continuously light-grown Euglena gracilis. FEBS Lett 40: 325-330 30. WINKLER U, W SAFrEL, H STABENAU 1982 Studies on the aminotransferases participating in the glycolate metabolism ofthe alga Mougeotia. Plant Physiol 70: 340-342 Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1985 American Society of Plant Biologists. All rights reserved.