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[25] Mitochondria from spinach leaves
[25]
MITOCHONDRIA FROM SPINACH LEAVES
133
are: (a) control of temperature (0-4 °) and pH (7.0-7.2); (b) avoidance of violent methods for the disruption of the tissue; (c) sedimentation of the washed mitochondria at 6000 g; and (d) careful separation of the mitochondrial fraction from other materials that sediment with the mitochondria.
[ 25 ] Mitochondria from Spinach Leaves
By I. ZELITCH 0hmura 1 first showed that mitochondria from spinach carried out oxidative phosphorylation. The following modified method produces mitochondria that give high P : 0 ratios with substrates of the citric acid cycle and DPNH. 2 Other studies on mitochondria isolated from green leaves have been described for tobacco, a
Preparation Spinach was purchased locally and kept at 5 ° in a plastic bag, sometimes for as long as several weeks, until used. Such storage had no effect on the activity of the isolated mitochondria. The younger pale-green leaves were taken, the midribs were excised, and a weighed quantity of leaf blade was washed in water and chilled on ice before being ground in the cold room. Grinding was done in a cold mortar with washed sand, with the addition of two volumes of grinding medium of the following composition: 0.45M sucrose, 0.05M mannit~l-borate buffer at pH 7.2, 0.03M potassium citrate, 0.01 M EDTA at pH 7.5, and 0.05 M tris(hydroxymethyl)aminomethane chloride (Tris) buffer at pH 8.3. The ground tissue, final pH of the suspension 7.2, was squeezed through two layers of cheesecloth and then centrifuged for 5 minutes at 600 g at 5 °. The residue, containing chloroplasts and other heavy cell fragments, was discarded, and the supernatant fluid was centrifuged for 20 minutes at 10,000 g. The residue, containing the mitochondria, was gently suspended in a washing medium (1 ml per gram of leaf) composed as follows: 0.3 M sucrose, 2 X 10-4 M EDTA, and 0.05 M Tris buffer at pH 7.5. The suspension was centrifuged for 20 minutes at 10,000 g. The mitochondria were finally taken up in the washing medium (1 ml per 1.8 g of leaf) for ' T . Ohmura, Arch. Biochem. Biophys. 57, 187 (1955). I. Zelitch and G. A. Barber, Plan~ Physiol. 35, 205 (1960). 3W. S. Pierpoint, Biochem. J. 71, 518 (1959). See also W. S. Pierpoint, Biochem. J. 82, 143 (1962).
134
PREPARATION OF MITOCHONDRIA
[25]
use in the experiments. Usually 0.9 ml of the suspension was placed in each Warburg vessel. This is equivalent to 1.6 g of original leaf tissue, and contained 0.5-0.8 mg of protein N, as measured by nesslerization after Kjeldahl digestion of the particles which had been washed with trichloroaeetic acid. The preparation was green in color, doubtless because of the presence of material originating in the chloroplasts. Oxidative Phosphorylation Oxygen uptake was measured by conventional techniques in 15-ml Warburg vessels in an air atmosphere at 30% Substrate, cofactors, and the Initochondria suspended in the washing medium were placed in the main compartment of chilled vessels. The center well contained KOH, and to the side arm glucose and an excess of hexokinase were added. A typical reaction mixture contained the following: sucrose, 300 micromoles; MgS04, 10 micromoles; substrate, 20 micromoles; potassium phosphate buffer at pH 7.0, 37 micromoles; yeast coenzyme concentrate, 1 mg; ATP, 2 micromoles; mitochondria in 0.9 ml of washing medium; and water to make the final volume 2.0 ml. In the side arm were glucose, 50 micromoles, and yeast hexokinase, 0.2 mg. After temperature equilibration for 5 minutes, at zero time, the contents of the side arm were tipped into the main compartment. At the end of the reaction period, usually 25 minutes, the vessels were quickly transferred to an ice bath and 1 ml of 3% perchloric acid was added to stop enzymatic reaction. After centrifugation of the reaction mixture, the orthophosphate remaining in the supernatant fluid was determined.4 Good agreement was obtained when the disappearance of orthophosphate was compared with the net increase in glucose 6-phosphate. In typical experiments, 2 the following P : 0 ratios were obtained: succinate, 2.1; fumarate, 2.8; L-malate, 2.6; pyruvate (in presence of a catalytic amount of malate), 2.7; DPNH, 2.5; glycolate, 0.1-0.3. Inhibitors The mitochondrial preparations described also oxidize glycolate, but there is negligible phosphorylation accompanying oxidation of this substrate. A competitive inhibitor of glycolate oxidase, a-hydroxy-2-pyridinemethanesulfonic acid, at 5 X 10-4M, inhibited the oxidation of glycolate 68%, but had no effect on the oxidation or phosphorylation with succinate. Azide, at 1 X 10-a M, inhibited succinate oxidation 67% and diminished phosphorylation, whereas oxygen uptake with glycolate 'K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926).
[26]
MITOCHONDRIA FROM YEAST
135
was enhanced. Thus the pathway of electron transport by the mitochondria is undoubtedly through the cytochrome system, except with glycolate when a flavoprotein oxidase is involved.
[ 26] Y e a s t M i t o c h o n d r i a a n d S u b m i t o c h o n d r i a l P a r t i c l e s By JAMES R. MATTOONand WALTERX. BALCAVAGE General Considerations
Mitochondria and submitochondrial particles have been prepared from several species of yeast, including Candida (Torulopsis) utilis, 1 Saccharomyces cerevisiae, 2,3 and closely related species such as S. carlsbergensis. 4 The foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria. Two approaches to this problem have been used: (1) Cells are ruptured mechanically and mitochondrial fragments are separated from more intact mitochondria by differential centrifugation and careful separation of supernatants and loosely packed (fluffy) layers (submitochondrial particles) from firmer pellets (mitochondria). (2) Yeast cells are subjected to enzymatic cell wall digestion in order to form osmotically sensitive spheroplasts (protoplasts), from which mitochondria are liberated by osmotic shock and mild homogenization. The main disadvantages of the mechanical rupture method are poor yields, the need for empirical selection of optimum breakage conditions, and the highly subjective process of fluffy layer separation. The enzymatic method, while giving good yields of reasonably intact mitochondria, requires careful control of enzyme source and/or digestion conditions to avoid premature lysis of spheroplasts. Since the most commonly used source of digestion enzymes, gut juice from the snail Helix pomatia, contains materials detrimental to mitochondrial activity (even after enzyme fractionation), careful washing of spheroplasts is required. The most critical problem encountered in the spheroplast method is the variation of cell wall sensitivity to digesting enzymes with different strains of yeast and with physiological state of the yeast. Stationary phase cells must be subjected to successive digestions in the 1A. E. 3 E. 4 T.
W. Linnane, E. Vitols, and P. Nowland, J. Ceil Biol. 13, 345 (1962). A. Duell, S. Inoue, and M. F. Utter, J. Bacteriol. 88, 1762 (1964). Vitols and A. W. Linnane, J. Biochem. Biophys. Cytol. 9, 701 (1961). Ohnishi and B. I-Iagihara, J. Biochem. (Tokyo) 55, 584 (1964).
MITOCHONDRIA FROM SPINACH LEAVES
133
are: (a) control of temperature (0-4 °) and pH (7.0-7.2); (b) avoidance of violent methods for the disruption of the tissue; (c) sedimentation of the washed mitochondria at 6000 g; and (d) careful separation of the mitochondrial fraction from other materials that sediment with the mitochondria.
[ 25 ] Mitochondria from Spinach Leaves
By I. ZELITCH 0hmura 1 first showed that mitochondria from spinach carried out oxidative phosphorylation. The following modified method produces mitochondria that give high P : 0 ratios with substrates of the citric acid cycle and DPNH. 2 Other studies on mitochondria isolated from green leaves have been described for tobacco, a
Preparation Spinach was purchased locally and kept at 5 ° in a plastic bag, sometimes for as long as several weeks, until used. Such storage had no effect on the activity of the isolated mitochondria. The younger pale-green leaves were taken, the midribs were excised, and a weighed quantity of leaf blade was washed in water and chilled on ice before being ground in the cold room. Grinding was done in a cold mortar with washed sand, with the addition of two volumes of grinding medium of the following composition: 0.45M sucrose, 0.05M mannit~l-borate buffer at pH 7.2, 0.03M potassium citrate, 0.01 M EDTA at pH 7.5, and 0.05 M tris(hydroxymethyl)aminomethane chloride (Tris) buffer at pH 8.3. The ground tissue, final pH of the suspension 7.2, was squeezed through two layers of cheesecloth and then centrifuged for 5 minutes at 600 g at 5 °. The residue, containing chloroplasts and other heavy cell fragments, was discarded, and the supernatant fluid was centrifuged for 20 minutes at 10,000 g. The residue, containing the mitochondria, was gently suspended in a washing medium (1 ml per gram of leaf) composed as follows: 0.3 M sucrose, 2 X 10-4 M EDTA, and 0.05 M Tris buffer at pH 7.5. The suspension was centrifuged for 20 minutes at 10,000 g. The mitochondria were finally taken up in the washing medium (1 ml per 1.8 g of leaf) for ' T . Ohmura, Arch. Biochem. Biophys. 57, 187 (1955). I. Zelitch and G. A. Barber, Plan~ Physiol. 35, 205 (1960). 3W. S. Pierpoint, Biochem. J. 71, 518 (1959). See also W. S. Pierpoint, Biochem. J. 82, 143 (1962).
134
PREPARATION OF MITOCHONDRIA
[25]
use in the experiments. Usually 0.9 ml of the suspension was placed in each Warburg vessel. This is equivalent to 1.6 g of original leaf tissue, and contained 0.5-0.8 mg of protein N, as measured by nesslerization after Kjeldahl digestion of the particles which had been washed with trichloroaeetic acid. The preparation was green in color, doubtless because of the presence of material originating in the chloroplasts. Oxidative Phosphorylation Oxygen uptake was measured by conventional techniques in 15-ml Warburg vessels in an air atmosphere at 30% Substrate, cofactors, and the Initochondria suspended in the washing medium were placed in the main compartment of chilled vessels. The center well contained KOH, and to the side arm glucose and an excess of hexokinase were added. A typical reaction mixture contained the following: sucrose, 300 micromoles; MgS04, 10 micromoles; substrate, 20 micromoles; potassium phosphate buffer at pH 7.0, 37 micromoles; yeast coenzyme concentrate, 1 mg; ATP, 2 micromoles; mitochondria in 0.9 ml of washing medium; and water to make the final volume 2.0 ml. In the side arm were glucose, 50 micromoles, and yeast hexokinase, 0.2 mg. After temperature equilibration for 5 minutes, at zero time, the contents of the side arm were tipped into the main compartment. At the end of the reaction period, usually 25 minutes, the vessels were quickly transferred to an ice bath and 1 ml of 3% perchloric acid was added to stop enzymatic reaction. After centrifugation of the reaction mixture, the orthophosphate remaining in the supernatant fluid was determined.4 Good agreement was obtained when the disappearance of orthophosphate was compared with the net increase in glucose 6-phosphate. In typical experiments, 2 the following P : 0 ratios were obtained: succinate, 2.1; fumarate, 2.8; L-malate, 2.6; pyruvate (in presence of a catalytic amount of malate), 2.7; DPNH, 2.5; glycolate, 0.1-0.3. Inhibitors The mitochondrial preparations described also oxidize glycolate, but there is negligible phosphorylation accompanying oxidation of this substrate. A competitive inhibitor of glycolate oxidase, a-hydroxy-2-pyridinemethanesulfonic acid, at 5 X 10-4M, inhibited the oxidation of glycolate 68%, but had no effect on the oxidation or phosphorylation with succinate. Azide, at 1 X 10-a M, inhibited succinate oxidation 67% and diminished phosphorylation, whereas oxygen uptake with glycolate 'K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926).
[26]
MITOCHONDRIA FROM YEAST
135
was enhanced. Thus the pathway of electron transport by the mitochondria is undoubtedly through the cytochrome system, except with glycolate when a flavoprotein oxidase is involved.
[ 26] Y e a s t M i t o c h o n d r i a a n d S u b m i t o c h o n d r i a l P a r t i c l e s By JAMES R. MATTOONand WALTERX. BALCAVAGE General Considerations
Mitochondria and submitochondrial particles have been prepared from several species of yeast, including Candida (Torulopsis) utilis, 1 Saccharomyces cerevisiae, 2,3 and closely related species such as S. carlsbergensis. 4 The foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria. Two approaches to this problem have been used: (1) Cells are ruptured mechanically and mitochondrial fragments are separated from more intact mitochondria by differential centrifugation and careful separation of supernatants and loosely packed (fluffy) layers (submitochondrial particles) from firmer pellets (mitochondria). (2) Yeast cells are subjected to enzymatic cell wall digestion in order to form osmotically sensitive spheroplasts (protoplasts), from which mitochondria are liberated by osmotic shock and mild homogenization. The main disadvantages of the mechanical rupture method are poor yields, the need for empirical selection of optimum breakage conditions, and the highly subjective process of fluffy layer separation. The enzymatic method, while giving good yields of reasonably intact mitochondria, requires careful control of enzyme source and/or digestion conditions to avoid premature lysis of spheroplasts. Since the most commonly used source of digestion enzymes, gut juice from the snail Helix pomatia, contains materials detrimental to mitochondrial activity (even after enzyme fractionation), careful washing of spheroplasts is required. The most critical problem encountered in the spheroplast method is the variation of cell wall sensitivity to digesting enzymes with different strains of yeast and with physiological state of the yeast. Stationary phase cells must be subjected to successive digestions in the 1A. E. 3 E. 4 T.
W. Linnane, E. Vitols, and P. Nowland, J. Ceil Biol. 13, 345 (1962). A. Duell, S. Inoue, and M. F. Utter, J. Bacteriol. 88, 1762 (1964). Vitols and A. W. Linnane, J. Biochem. Biophys. Cytol. 9, 701 (1961). Ohnishi and B. I-Iagihara, J. Biochem. (Tokyo) 55, 584 (1964).