- Email: [email protected]
[117] Isolation of ATP from muscle
862
COENZYMES AND RELATED PHOSPHATE COMPOUNDS
[117]
[117] Isolationof A T P from Muscle By Louis BERGER The method described is based mainly on the procedures of Lohmann and Schuster 1 and Kerr 2 as modified by Dounce et al. 3 ATP is extracted from muscle with TCA and carried through a series of precipitations as the mercury and barium salt. Probably any fresh muscle may be used, but for maximum yield and highest purity rabbit muscle is recommended. The anesthesia procedure of Du Bois et al., 4 described in step 1, is particularly recommended. Step 1. Isolation of the Muscle. Anesthetize a large male rabbit by a series of intraperitoneal injections of magnesium sulfate solution (51% MgSO4-7H20). The first dose is 1 ml./kg, of body weight followed by 0.5 ml./kg, every 10 minutes until complete anesthesia is reached. This takes about 30 minutes. Then decapitate the animal, and skin, eviscerate, and chill the carcass in ice water for 15 minutes. Dissect the muscle from any part of the carcass, and pass it through a chilled meat grinder. Not more than 30 minutes should be spent in dissecting, even if some of the muscle must be sacrificed. One large rabbit (4 kg.) will yield 0.8 to 1 kg. of muscle. Unless otherwise stated, all subsequent operations are performed at temperatures as near to 0 ° as is practical. Step 2. Extraction of the Muscle with TCA. Quickly weigh the muscle, and extract 200-g. portions for 2 minutes in a Waring blendor with an equal volume of 10% TCA. Squeeze the liquid through a double layer of cheesecloth and re-extract the residue as above, this time with 5 % TCA. Filter the combined turbid extracts by suction through paper, recycling until a clear filtrate is obtained. Adjust the pH of the filtrate to 7 with 10% NaOH. All subsequent steps will be described for a neutralized filtrate obtained from I kg. of muscle. If more than one rabbit is used, it is recommended that steps 1 and 2 be performed for each rabbit individually, and the filtrates pooled. Step 3. First Precipitation of Hg Salt of A TP. Slowly add 4 ml. of glacial acetic acid to the 2 1. of filtrate with stirring, followed by sufficient mercuric acetate reagent (20% mercuric acetate in 2% acetic acid) to 1 K. L o h m a n n a n d P. Schuster, Biochem. Z. 282, 104 (1935). 2 S. E. Kerr, J. Biol. Chem. 139, 12l (1941). A. L. Dounce, A. Rothstein, G. T. Beyer, R. Meier, a n d R. M. Freer, J. Biol. Chem. 174~ 361 (1948). 4 K. P. DuBois, H. G. Albaum, a n d V. R. Potter, J. Biol. Chem. 147, 699 (1943).
[117]
ISOLATION OF ATP FROM MUSCLE
863
give complete precipitation (150 to 200 ml. required). Allow the mercury salt to settle for 1 to 3 hours, after which time most of the supernatant fluid will be clear enough to decant. Collect the precipitate at the centrifuge, and wash it once with three times its volume of a 1:40 dilution of the mercuric acetate reagent. Step ~. First Precipitation of the Ba Salt of A TP. Suspend the washed precipitate in 400 ml. of water, and decompose the mercury salt with H~S. This is best accomplished in a closed system by applying vacuum to the container before attaching it to the H~S source. With continuous shaking the reaction will be complete in about 10 minutes, after which time no more H2S will be consumed. Filter the preparation by suction; recycle if necessary to obtain a clear filtrate. Wash the HgS cake three times with 20-ml. portions of cold water. Free the combined filtrates of H~S by passing air through the solution. About 1 hour is usually required. Adjust the pH of this solution to 7 with 10% NaOH. Add 20 ml. of 2 M barium acetate (an excess) to precipitate the barium salt of ATP. Allow the precipitate to settle for about 1 hour, after which time most of the supernatant fluid will be clear enough to decant. Collect the precipitate at the centrifuge, and wash it twice with three times its volume of cold water. Step 5. Second Precipitation of Hg Salt of A TP. Suspend the washed barium precipitate in 500 ml. of cold water, and add the minimum amount of glacial acetic acid to effect almost complete solution. A small amount of precipitate may remain which should be filtered or centrifuged off. The pH of the solution should not be allowed to go below 3. Add one-tenth the amount of mercuric acetate reagent required in step 3 (about 20 ml.), and collect and wash the mercury precipitate as in step 3. Step 6. Second Precipitation of Ba Salt of A TP. Decompose the washed mercury precipitate with H:S as in step 4, and obtain an aerated filtrate as described there. Slowly add 10% NaOH until a small amount of precipitate forms, keeping the pH below 6. The precipitate is barium ATP resulting from a small amount of barium which is carried through with the mercury precipitations. If no precipitate forms when pH 6 is reached, add 1 drop of 2 M barium acetate so as to allow a small amount of precipitate to form. Filter off the precipitate, which will remove colloidal sulfides. Adjust the pH of the clear filtrate to 7 with 10% NaOH, and precipitate the barium salt of ATP with barium acetate as described in step 4. Wash the precipitate at the centrifuge successively witti 100-ml. portions of the following: twice with cold water, once with 50% ethanol, once with 75% ethanol, twice with 95% ethanol, and twice with ether. Finally, air-dry the product and then place it in a vacuum desiccator over activated alumina or calcium chloride.
864
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[118]
Two to three grams of BasATP.4HsO 6 is obtained from 1 kg. of muscle. Chromatographic analysis 6 and enzymatic assay 7 usually indicate a p u r i t y of 90 to 95 %. The major i m p u r i t y is the barium salt of ADP. Usually less t h a n 1% of the total phosphorus is present as inorganic phosphorus. Preparations of A T P , virtually free of A D P , can be made from this product b y chromatographic separation, e s Commercially available from various sources as the Ba, Na, or K salt. e See Vol. III [120]. 7 See Vol. III [121].
[118]
Preparation o f A D P from A T P By L o u i s BERGER
A D P is prepared from A T P by enzymatic hydrolysis using the adenosirmtriphosphatase of lobster muscle. 1 R a b b i t muscle ~,3 may also be used as the source of the enzyme, but it is more difficult to prepare and is more likely to contain interfering enzymes2 Step 1. Preparation of the Lobster Muscle. Cut a live lobster in half, transversely, and remove the tail muscles. Cut t h e m into strips with scissors (do not mince or grind). Suspend the strips (25 to 35 g.) in 300 ml. of cold 0.45% KC1, and mechanically stir slowly for 15 minutes. Change the solution every 15 minutes until a total of five washings have been made. T h e strips are now ready for use in step 3. Step 2. Preparation of A TP Solution. Dissolve 1 g. of the sodium or potassium salt of A T P 4 in 100 ml. of water. If the barium salt of A T P 4.s is used, it must first be freed of barium as follows: dissolve 1.1 g. in 40 ml. of cold 0,1 N HC1, and add 0.3 g. of Na:SO4. Centrifuge the BaSO4, wash it once with 10 ml. of the cold acid, and again centrifuge. Neutralize the combined s u p e r n a t a n t fluids with 10% N a O H to p H 7, and dilute to 100 ml. with water. If the N a or Ba salt of A T P was used to prepare the solution, add solid KCI to make the solution 0.1 M in K ions. Step 3. Enzymatic Conversion of A TP to ADP. Add the washed muscle strips of step 1 to the 100 ml. of 1% A T P solution of step 2. Slowly stir I K. Lohmann, Biochem. Z. 282, 109 (1935). z K. Bailey, Biochem. J. 36, 121 (1942). 8 I. Green, J. R. C. Brown, and W. F. H. iV[. Mommaerts, J. Biol. Chem. 205, 493 (1953). 4 ADP is now commercially available from various sources. 5See Vol. III [117].
COENZYMES AND RELATED PHOSPHATE COMPOUNDS
[117]
[117] Isolationof A T P from Muscle By Louis BERGER The method described is based mainly on the procedures of Lohmann and Schuster 1 and Kerr 2 as modified by Dounce et al. 3 ATP is extracted from muscle with TCA and carried through a series of precipitations as the mercury and barium salt. Probably any fresh muscle may be used, but for maximum yield and highest purity rabbit muscle is recommended. The anesthesia procedure of Du Bois et al., 4 described in step 1, is particularly recommended. Step 1. Isolation of the Muscle. Anesthetize a large male rabbit by a series of intraperitoneal injections of magnesium sulfate solution (51% MgSO4-7H20). The first dose is 1 ml./kg, of body weight followed by 0.5 ml./kg, every 10 minutes until complete anesthesia is reached. This takes about 30 minutes. Then decapitate the animal, and skin, eviscerate, and chill the carcass in ice water for 15 minutes. Dissect the muscle from any part of the carcass, and pass it through a chilled meat grinder. Not more than 30 minutes should be spent in dissecting, even if some of the muscle must be sacrificed. One large rabbit (4 kg.) will yield 0.8 to 1 kg. of muscle. Unless otherwise stated, all subsequent operations are performed at temperatures as near to 0 ° as is practical. Step 2. Extraction of the Muscle with TCA. Quickly weigh the muscle, and extract 200-g. portions for 2 minutes in a Waring blendor with an equal volume of 10% TCA. Squeeze the liquid through a double layer of cheesecloth and re-extract the residue as above, this time with 5 % TCA. Filter the combined turbid extracts by suction through paper, recycling until a clear filtrate is obtained. Adjust the pH of the filtrate to 7 with 10% NaOH. All subsequent steps will be described for a neutralized filtrate obtained from I kg. of muscle. If more than one rabbit is used, it is recommended that steps 1 and 2 be performed for each rabbit individually, and the filtrates pooled. Step 3. First Precipitation of Hg Salt of A TP. Slowly add 4 ml. of glacial acetic acid to the 2 1. of filtrate with stirring, followed by sufficient mercuric acetate reagent (20% mercuric acetate in 2% acetic acid) to 1 K. L o h m a n n a n d P. Schuster, Biochem. Z. 282, 104 (1935). 2 S. E. Kerr, J. Biol. Chem. 139, 12l (1941). A. L. Dounce, A. Rothstein, G. T. Beyer, R. Meier, a n d R. M. Freer, J. Biol. Chem. 174~ 361 (1948). 4 K. P. DuBois, H. G. Albaum, a n d V. R. Potter, J. Biol. Chem. 147, 699 (1943).
[117]
ISOLATION OF ATP FROM MUSCLE
863
give complete precipitation (150 to 200 ml. required). Allow the mercury salt to settle for 1 to 3 hours, after which time most of the supernatant fluid will be clear enough to decant. Collect the precipitate at the centrifuge, and wash it once with three times its volume of a 1:40 dilution of the mercuric acetate reagent. Step ~. First Precipitation of the Ba Salt of A TP. Suspend the washed precipitate in 400 ml. of water, and decompose the mercury salt with H~S. This is best accomplished in a closed system by applying vacuum to the container before attaching it to the H~S source. With continuous shaking the reaction will be complete in about 10 minutes, after which time no more H2S will be consumed. Filter the preparation by suction; recycle if necessary to obtain a clear filtrate. Wash the HgS cake three times with 20-ml. portions of cold water. Free the combined filtrates of H~S by passing air through the solution. About 1 hour is usually required. Adjust the pH of this solution to 7 with 10% NaOH. Add 20 ml. of 2 M barium acetate (an excess) to precipitate the barium salt of ATP. Allow the precipitate to settle for about 1 hour, after which time most of the supernatant fluid will be clear enough to decant. Collect the precipitate at the centrifuge, and wash it twice with three times its volume of cold water. Step 5. Second Precipitation of Hg Salt of A TP. Suspend the washed barium precipitate in 500 ml. of cold water, and add the minimum amount of glacial acetic acid to effect almost complete solution. A small amount of precipitate may remain which should be filtered or centrifuged off. The pH of the solution should not be allowed to go below 3. Add one-tenth the amount of mercuric acetate reagent required in step 3 (about 20 ml.), and collect and wash the mercury precipitate as in step 3. Step 6. Second Precipitation of Ba Salt of A TP. Decompose the washed mercury precipitate with H:S as in step 4, and obtain an aerated filtrate as described there. Slowly add 10% NaOH until a small amount of precipitate forms, keeping the pH below 6. The precipitate is barium ATP resulting from a small amount of barium which is carried through with the mercury precipitations. If no precipitate forms when pH 6 is reached, add 1 drop of 2 M barium acetate so as to allow a small amount of precipitate to form. Filter off the precipitate, which will remove colloidal sulfides. Adjust the pH of the clear filtrate to 7 with 10% NaOH, and precipitate the barium salt of ATP with barium acetate as described in step 4. Wash the precipitate at the centrifuge successively witti 100-ml. portions of the following: twice with cold water, once with 50% ethanol, once with 75% ethanol, twice with 95% ethanol, and twice with ether. Finally, air-dry the product and then place it in a vacuum desiccator over activated alumina or calcium chloride.
864
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[118]
Two to three grams of BasATP.4HsO 6 is obtained from 1 kg. of muscle. Chromatographic analysis 6 and enzymatic assay 7 usually indicate a p u r i t y of 90 to 95 %. The major i m p u r i t y is the barium salt of ADP. Usually less t h a n 1% of the total phosphorus is present as inorganic phosphorus. Preparations of A T P , virtually free of A D P , can be made from this product b y chromatographic separation, e s Commercially available from various sources as the Ba, Na, or K salt. e See Vol. III [120]. 7 See Vol. III [121].
[118]
Preparation o f A D P from A T P By L o u i s BERGER
A D P is prepared from A T P by enzymatic hydrolysis using the adenosirmtriphosphatase of lobster muscle. 1 R a b b i t muscle ~,3 may also be used as the source of the enzyme, but it is more difficult to prepare and is more likely to contain interfering enzymes2 Step 1. Preparation of the Lobster Muscle. Cut a live lobster in half, transversely, and remove the tail muscles. Cut t h e m into strips with scissors (do not mince or grind). Suspend the strips (25 to 35 g.) in 300 ml. of cold 0.45% KC1, and mechanically stir slowly for 15 minutes. Change the solution every 15 minutes until a total of five washings have been made. T h e strips are now ready for use in step 3. Step 2. Preparation of A TP Solution. Dissolve 1 g. of the sodium or potassium salt of A T P 4 in 100 ml. of water. If the barium salt of A T P 4.s is used, it must first be freed of barium as follows: dissolve 1.1 g. in 40 ml. of cold 0,1 N HC1, and add 0.3 g. of Na:SO4. Centrifuge the BaSO4, wash it once with 10 ml. of the cold acid, and again centrifuge. Neutralize the combined s u p e r n a t a n t fluids with 10% N a O H to p H 7, and dilute to 100 ml. with water. If the N a or Ba salt of A T P was used to prepare the solution, add solid KCI to make the solution 0.1 M in K ions. Step 3. Enzymatic Conversion of A TP to ADP. Add the washed muscle strips of step 1 to the 100 ml. of 1% A T P solution of step 2. Slowly stir I K. Lohmann, Biochem. Z. 282, 109 (1935). z K. Bailey, Biochem. J. 36, 121 (1942). 8 I. Green, J. R. C. Brown, and W. F. H. iV[. Mommaerts, J. Biol. Chem. 205, 493 (1953). 4 ADP is now commercially available from various sources. 5See Vol. III [117].