Arch. Microbiol. 105, 187-192 (1975) - 9 by Springer-Verlag 1975
The Isolation and Characterization of Peroxisomes (Microbodies) from Baker's Yeast, Saccharomyces cerevisiae ROGER W. PARISH Cytological Laboratory, Institute of General Botany, University of Zfirich, Ztirich Received May 20, 1975 Abstract. Peroxisomes were isolated from derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0~ using glass beads). Catalase and urate oxidase, along with low activities of D-amino acid oxidase and e-c~-hydroxyacidoxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were
destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released or present in a labile second class ofperoxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a lowdensity particle as had been previously reported.
Key words: Peroxisomes (microbodies) - Saccharomyces cerevisiae - Catalase - Urate oxidase - Glyoxylate cycle.
Catalase activity in yeast is at least paritally sedimentable, suggesting an organelle association. Density gradient and zonal centrifugation have shown catalaserich particles (peroxisomes) with densities resembling mitochondria (/.19) (Perlman and Mahler, 1970) or somewhat lighter (1.169) (Szabo and Avers, 1969). Szabo and Avers (1969) describe three principle organelles in the cells; mitochondria, electron-dense granules and electron-transparent bodies. All three organetles were associated with the catalase-rich fraction following centrifugation. The authors suggested that catalase was associated with the electron dense granules. The cytochemical procedure used for catalase detection stains mitochondria in yeast, presumably due to cytochrome c peroxidase activity (Todd and Vigil, 1972). Catalase, although present, was not detected in association with microbodies (peroxisomes) in glucose or galactose grown yeast. There was some evidence of such organelles in glycerol grown cells. Hoffmann et al. (/970) do report diamino-benzidine oxidation product in peroxisomes from glucose grown yeast, although using a permanganate fixation procedure which Todd and Vigil (1972) found unsatisfactory. Szabo and Avers (1969) reported that enzymes of the glyoxylate cycle (malate synthetase, glycollate oxidase, and isocitrate lyase) in addition to catalase were present in yeast peroxisomes. Perlman and Mah-
ler (1970), on the other hand, found isocitrate lyase totally nonparticulate, and citrate synthetase in mitochondria and a "new" smaller and less dense particle. We have isolated peroxisomes from Saccharomyces cerevisiae in an endeavour to resolve these conflicting results and to further characterize the enzymes present. We compare results using homogenates from protoplasts and whole cells of glucose-repressed and derepressed yeast. Materials and Methods Cells. Baker's yeast, Saccharomyces cerevisiae, strain LBG H 1022, was grown in liquid culture containing salts, trace elements and vitamins with ammonium and peptone as nitrogen source. The carbon Source was either 5 ~ glucose or lactate. The media in Erlenmeyer flasks (500 ml) were inoculated with 2 x 105 cells/ml and incubated in an oscillating shaker at 27~C. Since urate oxidase is inducible in yeast with uric acid (Lee and Roush, 1964), 0.1 mg/ml uric acid was added to the cultures. Homogenization. Cells were mixed with SF medium (0.5 M sorbitol, 2.5~ Ficoll in 0.05 M Tris HC1, pH 7.4) (Parish, 1971) and glass beads (0.45mm 0) (1:2:4, v/v/v), precooled, and broken in a "Merkenschlager" for 5 sec at 4~ Whole cells were removed by centrifugation and rehomogenized. The procedure was repeated three times and the supernatants pooled (= homogenate). Preparation of Protoplasts. Protoplasts were prepared using helicase, and lysed according to Matile and Wiemken (1967). Density Gradient Centrifugation. The homogenate was centrifuged at 500 x g for 10 rain and the resulting supernatant at
hTTTTTt 20000 × g for 20 min. The sediment was resuspended in SF medium and layered onto SF medium-sucrose gradients (see figures for details)• Recovery of enzyme activities from gradients was always good (> 90~).
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Enzyme Assays. The enzymes catalase, glycollate oxidase, citrate synthetase, malate dehydrogenase, isocitrate lyase, malate synthetase, urate oxidase, succinate dehydrogenase, acid and alkaline phosphatase, dichlorophenolindophenol (DIP) diaphorase and protein were measured as previously described (Parish, 1971, 1972a,b,c). Adenylate kinase was determined according to Brdiczka et al. 0968) and a D-amino acid oxidase using D-methionine according to Marshall and Sokatch (1968); alcohol dehydrogenase as described by Boehringer AG (Mannheim).
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Electron Microscopy. Gradient fractions containing the peaks ofcataIase or malate dehydrogenase activities were pooled and diluted with SF medium containing 3070 sucrose and 1 glutaraldehyde. They were subsequently prepared for electronmicroscopy as previously described (Parish, t971).
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Aj/i x, Results and Discussion Sedimentability of Enzymes and Intactness of Organelles The homogenization procedure used for whole cells undoubtedly broke a considerable number of peroxisomes and mitochondria. Hence, values for enzyme sedimentability cannot give true indications of soluble and bound activities in vivo. Nevertheless, between 30 and 50 ~ of catalase activity (when present) was sedimentable, implying that many peroxisomes remained intact. Moreover, the peroxisomes and mitochondria remained largely intact during resuspension of the 20000 x g sediment and density gradient centrifugation. Some damage, however, was incurred by the mitochondrial outer membrane since up to 70 ~ of adenylate kinase, an enzyme sequestered between the inner and outer mitochondrial membranes (Brdiczka et al., 1968), remained at the top of gradients (Fig. 3). Malate dehydrogenase, succinate dehydrogenase and citrate synthetase, enzymes associated with the mitochondrial matrix, were not readily released from the mitochondria (Figs. 1 and 2). We were unable to detect any sedimentable isocitrate lyase activities, either from protoplasts or homogenized whole cells, although activity was present in the supernatant. We found no malate synthetase activity. Glucose Repressed Cells
Protoplasts. Protoplasts made from glucose repressed cells were homogenized in SF medium using a Sorvall Omnimixer. The homogenate was centrifuged at 500 x g for 20 rain and the entire supernatant layered onto gradients. Fig. 1 shows that malate dehydrogen-
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Fig. IA and B. Distribution of enzyme activities among fractions from a linear gradient [SF medium- 63 ~ (w/v) sucrose, on 0.4 ml of a 70~ (w/v) sucrose cushion; all solutions in 0.05 M Tris-HCl, pH 7.4] after centrifugation of the 20000 x g sediment isolated following homogenization of glucose repressed cells. Centrifugation was for 17 hrs at 39000 rpm in an SW 39 swing-out head using a Spinco L50 ultracentrifuge'. No catalase activity was detected. (A) A. . . . A, alcohol dehydrogenase; E]----.--~, DIP diaphorase; ©..... 0, citrate synthetase; 0 - - - - 0 , acid p-nitrophenylphosphatase (pH 5.0). (B) i - - - i , malate dehydrogenase; [22-.-.-[2], ATPase (pH 8.0, Mg + +); J ..... A, alkaline p-nitrophenylphosphatase (pH 8.5) ase and citrate synthetase were present and sedimented through density gradients. The results suggest that two populations of particles are present, both containing malate dehydrogenase, DIP diaphorase and ATPase, but the lighter population with significantly less citrate synthetase than the heavier. Some malate dehydrogenase activity remained near the top of the gradients, suggesting that some mitochondrial breakage had occurred. No catalase activity was present in the homogehates. Alcohol dehydrogenase, a soluble enzyme,
R. W. Parish: Yeast Peroxisomes
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The Isolation and Characterization of Peroxisomes (Microbodies) from Baker's Yeast, Saccharomyces cerevisiae ROGER W. PARISH Cytological Laboratory, Institute of General Botany, University of Zfirich, Ztirich Received May 20, 1975 Abstract. Peroxisomes were isolated from derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0~ using glass beads). Catalase and urate oxidase, along with low activities of D-amino acid oxidase and e-c~-hydroxyacidoxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were
destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released or present in a labile second class ofperoxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a lowdensity particle as had been previously reported.
Key words: Peroxisomes (microbodies) - Saccharomyces cerevisiae - Catalase - Urate oxidase - Glyoxylate cycle.
Catalase activity in yeast is at least paritally sedimentable, suggesting an organelle association. Density gradient and zonal centrifugation have shown catalaserich particles (peroxisomes) with densities resembling mitochondria (/.19) (Perlman and Mahler, 1970) or somewhat lighter (1.169) (Szabo and Avers, 1969). Szabo and Avers (1969) describe three principle organelles in the cells; mitochondria, electron-dense granules and electron-transparent bodies. All three organetles were associated with the catalase-rich fraction following centrifugation. The authors suggested that catalase was associated with the electron dense granules. The cytochemical procedure used for catalase detection stains mitochondria in yeast, presumably due to cytochrome c peroxidase activity (Todd and Vigil, 1972). Catalase, although present, was not detected in association with microbodies (peroxisomes) in glucose or galactose grown yeast. There was some evidence of such organelles in glycerol grown cells. Hoffmann et al. (/970) do report diamino-benzidine oxidation product in peroxisomes from glucose grown yeast, although using a permanganate fixation procedure which Todd and Vigil (1972) found unsatisfactory. Szabo and Avers (1969) reported that enzymes of the glyoxylate cycle (malate synthetase, glycollate oxidase, and isocitrate lyase) in addition to catalase were present in yeast peroxisomes. Perlman and Mah-
ler (1970), on the other hand, found isocitrate lyase totally nonparticulate, and citrate synthetase in mitochondria and a "new" smaller and less dense particle. We have isolated peroxisomes from Saccharomyces cerevisiae in an endeavour to resolve these conflicting results and to further characterize the enzymes present. We compare results using homogenates from protoplasts and whole cells of glucose-repressed and derepressed yeast. Materials and Methods Cells. Baker's yeast, Saccharomyces cerevisiae, strain LBG H 1022, was grown in liquid culture containing salts, trace elements and vitamins with ammonium and peptone as nitrogen source. The carbon Source was either 5 ~ glucose or lactate. The media in Erlenmeyer flasks (500 ml) were inoculated with 2 x 105 cells/ml and incubated in an oscillating shaker at 27~C. Since urate oxidase is inducible in yeast with uric acid (Lee and Roush, 1964), 0.1 mg/ml uric acid was added to the cultures. Homogenization. Cells were mixed with SF medium (0.5 M sorbitol, 2.5~ Ficoll in 0.05 M Tris HC1, pH 7.4) (Parish, 1971) and glass beads (0.45mm 0) (1:2:4, v/v/v), precooled, and broken in a "Merkenschlager" for 5 sec at 4~ Whole cells were removed by centrifugation and rehomogenized. The procedure was repeated three times and the supernatants pooled (= homogenate). Preparation of Protoplasts. Protoplasts were prepared using helicase, and lysed according to Matile and Wiemken (1967). Density Gradient Centrifugation. The homogenate was centrifuged at 500 x g for 10 rain and the resulting supernatant at
hTTTTTt 20000 × g for 20 min. The sediment was resuspended in SF medium and layered onto SF medium-sucrose gradients (see figures for details)• Recovery of enzyme activities from gradients was always good (> 90~).
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Enzyme Assays. The enzymes catalase, glycollate oxidase, citrate synthetase, malate dehydrogenase, isocitrate lyase, malate synthetase, urate oxidase, succinate dehydrogenase, acid and alkaline phosphatase, dichlorophenolindophenol (DIP) diaphorase and protein were measured as previously described (Parish, 1971, 1972a,b,c). Adenylate kinase was determined according to Brdiczka et al. 0968) and a D-amino acid oxidase using D-methionine according to Marshall and Sokatch (1968); alcohol dehydrogenase as described by Boehringer AG (Mannheim).
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Aj/i x, Results and Discussion Sedimentability of Enzymes and Intactness of Organelles The homogenization procedure used for whole cells undoubtedly broke a considerable number of peroxisomes and mitochondria. Hence, values for enzyme sedimentability cannot give true indications of soluble and bound activities in vivo. Nevertheless, between 30 and 50 ~ of catalase activity (when present) was sedimentable, implying that many peroxisomes remained intact. Moreover, the peroxisomes and mitochondria remained largely intact during resuspension of the 20000 x g sediment and density gradient centrifugation. Some damage, however, was incurred by the mitochondrial outer membrane since up to 70 ~ of adenylate kinase, an enzyme sequestered between the inner and outer mitochondrial membranes (Brdiczka et al., 1968), remained at the top of gradients (Fig. 3). Malate dehydrogenase, succinate dehydrogenase and citrate synthetase, enzymes associated with the mitochondrial matrix, were not readily released from the mitochondria (Figs. 1 and 2). We were unable to detect any sedimentable isocitrate lyase activities, either from protoplasts or homogenized whole cells, although activity was present in the supernatant. We found no malate synthetase activity. Glucose Repressed Cells
Protoplasts. Protoplasts made from glucose repressed cells were homogenized in SF medium using a Sorvall Omnimixer. The homogenate was centrifuged at 500 x g for 20 rain and the entire supernatant layered onto gradients. Fig. 1 shows that malate dehydrogen-
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Fig. IA and B. Distribution of enzyme activities among fractions from a linear gradient [SF medium- 63 ~ (w/v) sucrose, on 0.4 ml of a 70~ (w/v) sucrose cushion; all solutions in 0.05 M Tris-HCl, pH 7.4] after centrifugation of the 20000 x g sediment isolated following homogenization of glucose repressed cells. Centrifugation was for 17 hrs at 39000 rpm in an SW 39 swing-out head using a Spinco L50 ultracentrifuge'. No catalase activity was detected. (A) A. . . . A, alcohol dehydrogenase; E]----.--~, DIP diaphorase; ©..... 0, citrate synthetase; 0 - - - - 0 , acid p-nitrophenylphosphatase (pH 5.0). (B) i - - - i , malate dehydrogenase; [22-.-.-[2], ATPase (pH 8.0, Mg + +); J ..... A, alkaline p-nitrophenylphosphatase (pH 8.5) ase and citrate synthetase were present and sedimented through density gradients. The results suggest that two populations of particles are present, both containing malate dehydrogenase, DIP diaphorase and ATPase, but the lighter population with significantly less citrate synthetase than the heavier. Some malate dehydrogenase activity remained near the top of the gradients, suggesting that some mitochondrial breakage had occurred. No catalase activity was present in the homogehates. Alcohol dehydrogenase, a soluble enzyme,
R. W. Parish: Yeast Peroxisomes
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