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[35] A-particles1 and P-particles2
194
PREPARATION OF SUBMITOCHONDRIAL PARTICLES
[35]
exogenous NAD through, presumably, a reversal of the reactions of oxidative phosphorylation. 17 The submitochondrial particle also has the advantage of being able to be produced with varying, and within limits predictable, phosphorylative capacities. This may be seen in the table, in which the P : 0 ratios of the various particles described above are tabulated, the energy at the three phosphorylation sites being generated and assayed as described in the assay section above. ~7H. LSw, H. Krueger, and D. M. Ziegler, Biochem. Biophys. Res. Commun. 5, 231
(1961).
[ 35] A - P a r t i c l e s 1 a n d P - P a r t i c l e s 2
By
JUNE M . FESSENDEN 2a a n d EFRAIM RACKER 2a P~ + A D P
Apo-partlcles • ATP coupling factors
A partial resolution of oxidative phosphorylation in submitochondrial particles can be achieved by a variety of physical, chemical, and enzymatic degradation procedures. Such apo-particles catalyze oxidation of D P N H or succinate with little accompanying ATP formation. The oxidation can be coupled to phosphorylation by the addition of specific proteins (coupling factors) isolated from mitochondria. Among the most useful and reproducible preparations of apo-particles are the A-particles and P-particles which are stimulated by coupling factor 1,~ coupling factor 2,4 coupling factor 3 ° and coupling factor 4 ~a (F1, F2, Fs, and F4, respectively). Assay Method
Principle. Deficient particles are used which catalyze oxidation but negligible phosphorylation. On addition of multiple coupling factors to these particles, a marked stimulation of phosphorylation is observed. Reagents Potassium phosphate, 0.3 M, pH 7.4 ATP, 0.1 M, pH 7.4 1j. M. Fessenden and E. Racker, J. Biol. Chem. 241, 2483 (1966). 2 T. E. Conover, R. L. Prairie, and E. Racker, J. Biol. Chem. 238, 2831 (1963). 2~This manuscript was prepared while the authors were at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. 3 See this volume [82]. See this volume [84a]. 5See this volume [84b]. 5a See this volume [85].
[35]
A-PARTICLES AND P-PARTICLES
195
MgS04, 0.1 M Sodium succinate, 0.5 M, pH 7.4 F1 (1 mK/ml) 3,e F= (1 mg/ml) 4 in 0.3 M potassium phosphate pH 7.4 F8 (10 mg/ml) 5 or F4 (10 mg/ml). 5a The 12-13% ammonium sulfate fraction is dialyzed 50 hours aga!nst 200 volumes of 0.25 M sucrose-0.01 M Tris-5 mM EDTA, pH 10.7. Deficient particles (25 mg/ml) Hexokinase (Boehringer) dialyzed overnight against 400 volumes of 5 mM EDTA, pH 7.4-1% glucose (500 units/ml) Tris-S04, 1.0M, pH 8.0 MgS04, 1.0 M ATP, 0.2 M, pH 7.4 Glucose, 1.0 M EDTA, 0.24 M, pH 7.4 Sucrose, 1.0 M Bovine serum albumin, 20% (crystalline, Armour Co.) dialyzed overnight against 100 volumes of 0.01 M Tris-S04, pH 7.4 Recrystallized 32Pi (2 X 106 cpm/ml) 7 Trichloroacetic acid, 50% Preparation o] Stock Solution. A mixture of 0.2 ml of hexokinase, 0.05 ml of Tris-S04, 0.02 ml of 1.0M MgS04, 0.05 ml of 0.2 M ATP, 0.32 ml of glucose, 0.02 ml of EDTA, 0.05 ml of bovine serum albumin, and 0.29 ml of sucrose is prepared and stored at --20 °. Procedure. To the main compartment of two Warburg vessels 0.02 ml of 0.1 M ATP, 0.02 ml of 0.1 M MgSO4, and 0.02 ml of deficient particles are pipetted. To one of the vessels 0.1 ml potassium phosphate, and to the other vessel 0.04 ml of F1, 0.1 ml of F~ (which contains potassium phosphate), and 0.02 ml of F4 or 0.03 ml of F3 are added in a volume of 0.38 ml. After incubation at 23 ° for 5 minutes, the vessels are put in ice and 0.05 ml of the stock solution, 0.02 ml of succinate, and 0.05 ml of 32p~ are added. The side arm of the vessel contains 0.05 ml of 50% trichloroacetic acid. The vessels are placed on manometers and equilibrated foI 51/~ minutes at 30 °. The stopcock is then closed, and the first reading is taken at 6 minutes. Subsequent readings are taken at 6-minute intervals
6Protein determinations for F1 are measured spectrophotometrically [0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941)], and for all others by the biuret procedure [(E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. 223, 147 (1956)]. 7See this volume [4].
196
PREPARATION OF SUBMITOCHONDRIAL PARTICLES
[35]
for 24 or 30 minutes. The initial oxygen uptake for the first 6 minutes is determined by extrapolation of the observed rate. The reaction is terminated by tipping in the contents of the side arm. The precipitate is centrifuged out, and 0.1 ml of the supernatant solution is extracted with isobutanol-benzene, s and 1.0 ml of the water layer is plated, dried, and counted in a Nuclear-Chicago flow counter. Preparation of Ammonia Particles (A-Particles) A 2.5-g sample of heavy layer beef heart mitochondria 9 is suspended in a final volume of 120 ml containing 0.15 ml of 0.5M EDTA, pH 7.4, and 22 ml of 0.25 M sucrose. The pH of the solution is adjusted to 9.2 with a freshly diluted solution of 1 N ammonium hydroxide. The suspension is exposed to sonic oscillation in 30-ml batches for 2 minutes in a Raytheon sonic oscillator (250 watts, 10 ke) cooled by flowing ice water. The suspension is centrifuged at 26,000 g for 10 minutes, and the pellet is discarded. The supernatant fluid is recentrifuged at 104,000 g for 60 minutes, and the supernatant solution is discarded. The pellet is homogenized in 80 ml of 0.25 M sucrose-1 mM EDTA, pH 7.5, and centrifuged at 104,000g for 30 minutes. The pellet is again homogenized with 80 ml of 0.25 M sucrose and centrifuged as above. The final pellet is homogenized in 0.25 M sucrose at a protein concentration of about 25 mg/ml and stored at --55 °. Preparation of Phosphatide Suspension Five grams of asolectin 2 are homogenized in 50 ml of a solution containing 0.25 M sucrose, 0.01 M Tris-S04, p H 7.4, 0.5 mM EDTA, pH 7.4, and 0.01 M thioglycerol, then exposed to sonic oscillation for 30 minutes in a Raytheon (250 watts, 10 kc) sonic oscillator cooled by flowing ice water. The suspension is dialyzed 12-18 hours against several changes of 10 volumes of the above buffer. The dialyzed suspension is centrifuged at 104,000 g for 60 minutes, and the pellet is discarded. The supernatant solution is flushed with nitrogen and stored in the refrigerator. This 10% asolectin preparation contains 3.25 mg of esterified phosphate per milliliter. It should be used as fresh as possible, since bacteria grow in this solution even at 2 ° . Preparation of P-Particles One gram of heavy layer beef heart mitochondria 9 is mixed with 0.4 ml of 0.5M EDTA, pH 7.4, 10 ml of 1.0M sucrose, and 8 ml of asolectin in a final volume of 40 ml. This suspension is exposed to sonic 8 See this volume [9].
° See Vol. VI [58].
[36]
STABLE PHOSPHORYLATING
PARTICLES FROM YEAST
197
oscillation in 20-ml batches in a Raytheon (250 watts, 10 kc) sonic oscillator for 2 minutes. The suspension is centrifuged at 26,000 g for 10 minutes. The pellet is discarded, and the supernatant fluid is recentrifuged at 104,000 g for 60 minutes. The supernatant solution is discarded, and the pellet is homogenized with 36 ml of 0.25 M sucrose-1 mM E D T A - I % asolectin. The suspension is centrifuged at 104,000g for 60 minutes, and the supernatant solution is discarded. The pellet is homogenized in 36 ml 0.25 M sucrose-1 mM EDTA and centrifuged for 60 minutes at 104,000 g. The supernatant solution is again discarded, and the pellet is homogenized in 0.25 M sucrose to a final protein concentration of about 25 mg/ml and stored at --55 °.
[ 36] S t a b l e P h o s p h o r y l a t i n g S u b m i t o c h o n d r i a l P a r t . i d e s f r o m Bakers' Yeast
By
GOTTFRIED SCHATZ 1
Preparation TM Packed, commercially grown (National Yeast Corp., Belleville, New Jersey) bakers' yeast cells, 681 g (1.5 pounds), are washed three times with distilled water and once with 0.25 M mannitol-20 mM Tris-S04, pH 7.4,-1 mM EDTA (MTE medium). For each washing step, the cells are suspended in the washing medium to a final volume of approximately 3 liters and are reisolated by centrifugation (15 minutes at 1300g). The packed pellet of washed cells is then stored at -~4 ° overnight. From this point on all operations are carried out in the cold. For homogenization, the cells are evenly suspended in MTE medium to a final volume of 960 ml, and 20-28 ml aliquots (see below) are shaken for 20 seconds with exactly 50 g of glass beads (diameter 0.45~0.50 ram; Bronwill Scientific Division, Rochester, New York) at 4000 cycles per minute in the Braun MSK Mechanical Cell Homogenizer (Bronwill Scientific Division), using the 75-ml glass homogenizing flasks. During the shaking, the temperature within the flasks is maintained below ~ 4 ° by cooling with liquid carbon dioxide. In order to obtain satisfactory (25-40%) cell breakage it is of great importance to fill the flasks to not more than two-thirds of capacity. The amount of cell suspension added has to be adjusted accordingly. The contents of each flask are shaken, then transferred to a chilled beaker. The flask is rinsed with approximately 20 ml of MTE medium, and the washing is added to the already homogenized cell suspension. The flask 1 See footnote 1, page 30. 1. G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966).
PREPARATION OF SUBMITOCHONDRIAL PARTICLES
[35]
exogenous NAD through, presumably, a reversal of the reactions of oxidative phosphorylation. 17 The submitochondrial particle also has the advantage of being able to be produced with varying, and within limits predictable, phosphorylative capacities. This may be seen in the table, in which the P : 0 ratios of the various particles described above are tabulated, the energy at the three phosphorylation sites being generated and assayed as described in the assay section above. ~7H. LSw, H. Krueger, and D. M. Ziegler, Biochem. Biophys. Res. Commun. 5, 231
(1961).
[ 35] A - P a r t i c l e s 1 a n d P - P a r t i c l e s 2
By
JUNE M . FESSENDEN 2a a n d EFRAIM RACKER 2a P~ + A D P
Apo-partlcles • ATP coupling factors
A partial resolution of oxidative phosphorylation in submitochondrial particles can be achieved by a variety of physical, chemical, and enzymatic degradation procedures. Such apo-particles catalyze oxidation of D P N H or succinate with little accompanying ATP formation. The oxidation can be coupled to phosphorylation by the addition of specific proteins (coupling factors) isolated from mitochondria. Among the most useful and reproducible preparations of apo-particles are the A-particles and P-particles which are stimulated by coupling factor 1,~ coupling factor 2,4 coupling factor 3 ° and coupling factor 4 ~a (F1, F2, Fs, and F4, respectively). Assay Method
Principle. Deficient particles are used which catalyze oxidation but negligible phosphorylation. On addition of multiple coupling factors to these particles, a marked stimulation of phosphorylation is observed. Reagents Potassium phosphate, 0.3 M, pH 7.4 ATP, 0.1 M, pH 7.4 1j. M. Fessenden and E. Racker, J. Biol. Chem. 241, 2483 (1966). 2 T. E. Conover, R. L. Prairie, and E. Racker, J. Biol. Chem. 238, 2831 (1963). 2~This manuscript was prepared while the authors were at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. 3 See this volume [82]. See this volume [84a]. 5See this volume [84b]. 5a See this volume [85].
[35]
A-PARTICLES AND P-PARTICLES
195
MgS04, 0.1 M Sodium succinate, 0.5 M, pH 7.4 F1 (1 mK/ml) 3,e F= (1 mg/ml) 4 in 0.3 M potassium phosphate pH 7.4 F8 (10 mg/ml) 5 or F4 (10 mg/ml). 5a The 12-13% ammonium sulfate fraction is dialyzed 50 hours aga!nst 200 volumes of 0.25 M sucrose-0.01 M Tris-5 mM EDTA, pH 10.7. Deficient particles (25 mg/ml) Hexokinase (Boehringer) dialyzed overnight against 400 volumes of 5 mM EDTA, pH 7.4-1% glucose (500 units/ml) Tris-S04, 1.0M, pH 8.0 MgS04, 1.0 M ATP, 0.2 M, pH 7.4 Glucose, 1.0 M EDTA, 0.24 M, pH 7.4 Sucrose, 1.0 M Bovine serum albumin, 20% (crystalline, Armour Co.) dialyzed overnight against 100 volumes of 0.01 M Tris-S04, pH 7.4 Recrystallized 32Pi (2 X 106 cpm/ml) 7 Trichloroacetic acid, 50% Preparation o] Stock Solution. A mixture of 0.2 ml of hexokinase, 0.05 ml of Tris-S04, 0.02 ml of 1.0M MgS04, 0.05 ml of 0.2 M ATP, 0.32 ml of glucose, 0.02 ml of EDTA, 0.05 ml of bovine serum albumin, and 0.29 ml of sucrose is prepared and stored at --20 °. Procedure. To the main compartment of two Warburg vessels 0.02 ml of 0.1 M ATP, 0.02 ml of 0.1 M MgSO4, and 0.02 ml of deficient particles are pipetted. To one of the vessels 0.1 ml potassium phosphate, and to the other vessel 0.04 ml of F1, 0.1 ml of F~ (which contains potassium phosphate), and 0.02 ml of F4 or 0.03 ml of F3 are added in a volume of 0.38 ml. After incubation at 23 ° for 5 minutes, the vessels are put in ice and 0.05 ml of the stock solution, 0.02 ml of succinate, and 0.05 ml of 32p~ are added. The side arm of the vessel contains 0.05 ml of 50% trichloroacetic acid. The vessels are placed on manometers and equilibrated foI 51/~ minutes at 30 °. The stopcock is then closed, and the first reading is taken at 6 minutes. Subsequent readings are taken at 6-minute intervals
6Protein determinations for F1 are measured spectrophotometrically [0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941)], and for all others by the biuret procedure [(E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. 223, 147 (1956)]. 7See this volume [4].
196
PREPARATION OF SUBMITOCHONDRIAL PARTICLES
[35]
for 24 or 30 minutes. The initial oxygen uptake for the first 6 minutes is determined by extrapolation of the observed rate. The reaction is terminated by tipping in the contents of the side arm. The precipitate is centrifuged out, and 0.1 ml of the supernatant solution is extracted with isobutanol-benzene, s and 1.0 ml of the water layer is plated, dried, and counted in a Nuclear-Chicago flow counter. Preparation of Ammonia Particles (A-Particles) A 2.5-g sample of heavy layer beef heart mitochondria 9 is suspended in a final volume of 120 ml containing 0.15 ml of 0.5M EDTA, pH 7.4, and 22 ml of 0.25 M sucrose. The pH of the solution is adjusted to 9.2 with a freshly diluted solution of 1 N ammonium hydroxide. The suspension is exposed to sonic oscillation in 30-ml batches for 2 minutes in a Raytheon sonic oscillator (250 watts, 10 ke) cooled by flowing ice water. The suspension is centrifuged at 26,000 g for 10 minutes, and the pellet is discarded. The supernatant fluid is recentrifuged at 104,000 g for 60 minutes, and the supernatant solution is discarded. The pellet is homogenized in 80 ml of 0.25 M sucrose-1 mM EDTA, pH 7.5, and centrifuged at 104,000g for 30 minutes. The pellet is again homogenized with 80 ml of 0.25 M sucrose and centrifuged as above. The final pellet is homogenized in 0.25 M sucrose at a protein concentration of about 25 mg/ml and stored at --55 °. Preparation of Phosphatide Suspension Five grams of asolectin 2 are homogenized in 50 ml of a solution containing 0.25 M sucrose, 0.01 M Tris-S04, p H 7.4, 0.5 mM EDTA, pH 7.4, and 0.01 M thioglycerol, then exposed to sonic oscillation for 30 minutes in a Raytheon (250 watts, 10 kc) sonic oscillator cooled by flowing ice water. The suspension is dialyzed 12-18 hours against several changes of 10 volumes of the above buffer. The dialyzed suspension is centrifuged at 104,000 g for 60 minutes, and the pellet is discarded. The supernatant solution is flushed with nitrogen and stored in the refrigerator. This 10% asolectin preparation contains 3.25 mg of esterified phosphate per milliliter. It should be used as fresh as possible, since bacteria grow in this solution even at 2 ° . Preparation of P-Particles One gram of heavy layer beef heart mitochondria 9 is mixed with 0.4 ml of 0.5M EDTA, pH 7.4, 10 ml of 1.0M sucrose, and 8 ml of asolectin in a final volume of 40 ml. This suspension is exposed to sonic 8 See this volume [9].
° See Vol. VI [58].
[36]
STABLE PHOSPHORYLATING
PARTICLES FROM YEAST
197
oscillation in 20-ml batches in a Raytheon (250 watts, 10 kc) sonic oscillator for 2 minutes. The suspension is centrifuged at 26,000 g for 10 minutes. The pellet is discarded, and the supernatant fluid is recentrifuged at 104,000 g for 60 minutes. The supernatant solution is discarded, and the pellet is homogenized with 36 ml of 0.25 M sucrose-1 mM E D T A - I % asolectin. The suspension is centrifuged at 104,000g for 60 minutes, and the supernatant solution is discarded. The pellet is homogenized in 36 ml 0.25 M sucrose-1 mM EDTA and centrifuged for 60 minutes at 104,000 g. The supernatant solution is again discarded, and the pellet is homogenized in 0.25 M sucrose to a final protein concentration of about 25 mg/ml and stored at --55 °.
[ 36] S t a b l e P h o s p h o r y l a t i n g S u b m i t o c h o n d r i a l P a r t . i d e s f r o m Bakers' Yeast
By
GOTTFRIED SCHATZ 1
Preparation TM Packed, commercially grown (National Yeast Corp., Belleville, New Jersey) bakers' yeast cells, 681 g (1.5 pounds), are washed three times with distilled water and once with 0.25 M mannitol-20 mM Tris-S04, pH 7.4,-1 mM EDTA (MTE medium). For each washing step, the cells are suspended in the washing medium to a final volume of approximately 3 liters and are reisolated by centrifugation (15 minutes at 1300g). The packed pellet of washed cells is then stored at -~4 ° overnight. From this point on all operations are carried out in the cold. For homogenization, the cells are evenly suspended in MTE medium to a final volume of 960 ml, and 20-28 ml aliquots (see below) are shaken for 20 seconds with exactly 50 g of glass beads (diameter 0.45~0.50 ram; Bronwill Scientific Division, Rochester, New York) at 4000 cycles per minute in the Braun MSK Mechanical Cell Homogenizer (Bronwill Scientific Division), using the 75-ml glass homogenizing flasks. During the shaking, the temperature within the flasks is maintained below ~ 4 ° by cooling with liquid carbon dioxide. In order to obtain satisfactory (25-40%) cell breakage it is of great importance to fill the flasks to not more than two-thirds of capacity. The amount of cell suspension added has to be adjusted accordingly. The contents of each flask are shaken, then transferred to a chilled beaker. The flask is rinsed with approximately 20 ml of MTE medium, and the washing is added to the already homogenized cell suspension. The flask 1 See footnote 1, page 30. 1. G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966).