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Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity
ANALYTICAL
BIOCHEMISTRY
Three-Step
137, 374-379
Preparation and Purification of Phosphorus-33-Labeled Creatine Phosphate of High Specific Activity F. SAVABI,
lfniversit.v
(I 984)
of Southern
Ca@rnia
P. J. GEIGER,
School
r$ MedicGe, Received
AND S. P. BESSMAN 2025 Zonal.4venue,
August
Los Angeles,
Califbrnia
90033
5, 1983
Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P,) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P, is described. KEY WORDS: creatine phosphate: synthesis; inorganic phosphate removal: radioactive phosphate.
Radioautography studies of the creatine phosphate (CP)’ energy shuttle (1) required phosphorus-labeledcreatine phosphate of high purity to record the movements of the phosphate group during the contraction process. 33Pihas much too high energy (max 1710 kV) for electron micrographic studies, but it appears possible to use 33Pias a label because its energy (max 248 kV) is lessthan twice as high as 14C(max 156 kV). The design of the experiment required as little contamination as possible with labeled adenine nucleotides. The synthesis of radioactive creatine phosphate used in biochemical reactions has been carried out with purified creatine phosphokinase (EC 2.7.3.2) and a system for regenerating labeled ATP usually with a final yield of lessthan 25% based on radioactive phosphate (2). Phosphorus-labeled creatine phosphate hasalsobeen synthesized from y-labeled ATP, prepared with synthetic labeled carbamyl-P (3-6). The yield of ATP in this case, before several steps of purification, is about 75% on the basis of Pi. Yagi and Noda in ’ Abbreviations used: CP, creatine phosphate: creatine phosphokinase; CP 33. ‘jP-labeled creatine phate. 0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
CPK, phos-
374
1960 (7) used liver mitochondria for the synthesis of [T-~~P]ATP. The synthesized [y32P]ATP was then purified and used for the enzymatic synthesisof 32P-creatinephosphate. The final purified 32P-creatinephosphate still contained 5% Pi and the overall yield was 36% on the basis of 32Pi.We present here a highyield simplified procedure for direct mitochondrial synthesisof 33P,-creatinephosphate. Our method is based on the coupled reaction of mitochondrial oxidative phosphorylation and mitochondrial-bound creatine phosphokinase (CPK) (8- I I), using rabbit heart mitochondria, excesscreatine, inorganic 33Piphosphate, and 50-70 PM ADP. MATERIALS Male
New Zealand
AND white
METHODS rabbits,
6-7
lb.,
were obtained from ABC Rabbitry, Rosemead, California. Trizma base, creatine, nucleotides, dithiothreitol (DTT), and EDTA were purchased from Sigma Chemical, St. Louis, Missouri. Alamine(tri-n-octyl tertiaryamine) was purchased from McKerson Corporation, Minneapolis, Minnesota. FreonTP was obtained from Miller-Stephenson Company, Danbury. Connecticut. Beta-Phase
33P-CREATINE
PHOSPHATE
scintillation1 cocktail was purchased from WestChem Products, Inc., San Diego, California. Isotope 33P-H3P04 was obtained from New England Nuclear (Boston, Mass.). Activated charcoal was obtained from Matheson, Coleman and Bell, Cincinnati, Ohio.
Preparation
qf rabbit heart mitochondria.
Mitochondria were isolated from the ventricles of New Zealand albino rabbits according to the method of Yang et al. ( 1977) (12) with substitution of Tris-Cl for Tris-PO4 in all solutions. The homogenization medium contained 0.35 ‘M mannitol, 0.1 mM EDTA, and 10 mM T&-Cl (pH 7.3). Suspending medium contained. in addition to the above, 0.5% dialyzed bovine serum albumin. Polarographic studies were carried out on all preparations and only those having respiratory control ratios higher than 11 in the absence of Mg2+ were used.
Incubation conditions and protocol,for creatine phosphate synthesis. To optimize the conditions for the synthesis of creatine phosphate, several variables, including concentration of inorganic phosphate, ADP, mitochondrial protein, and different incubation times, were tested. The 1.582-ml (sum of additions) reaction mixture contained 0.5 ml “cocktail” [consisting of 0.543 M mannitol, 0.2 17 mM EDTA, 2 1.5’ mM Tris-Cl (pH 7.2) 3 1.7 mM KCI, 0.3 17 mM DTT, 6.34 mM MgCl?, and different concentrations of potassium phosphate buffer (pH 7.2)], 0.5 ml Hz0 or 33Pi solution, 0 to 0.3 ml suspending medium, 0.5 to 0.2 ml mitochondrial suspension. 7 ~1 of ADP solution, 75 ~1 of 0.1 M cu-ketoglutarate, and 5.2 mg of crystalline creatine. The final reaction medium contained 0.25 M mannitol, 10 mM KCl, 10 mM Tris-Cl (pH 7.2). 0.1 mM EDTA, 0.1 mM DTT, 2 mM MgC12, 25 mM creatine, 5 mM a-ketoglutarate, and various concentrations of either ADP, Pi. or mitochondrial protein. The creatine was added to 0.5 ml cocktail in about 15 X loo-mm plastic tube., 0.5 ml Hz0 plus or minus 3’Pi was then added. and the mixture was shaken until the creatine was dissolved. ADP and (Yketoglutarate were then added and the reaction
SYNTHESIS
375
was started by addition of mitochondrial suspension. The open test tube containing this mixture was then shaken at 3/s at 20°C. The reaction was stopped by addition of 1 ml of 1.4 M HC104 and centrifuged at 3000 rpm for 10 min. The supernatant solution was decanted carefully. At this point unlabeled ATP and ADP were added to the supernatant solution to final 3 and 2 mM concentrations, respectively, to dilute the radioactive nucleotides. About 55 mg activated charcoal was then added to the solution, which was centrifuged at 3000 r-pm for 5 min ( 13). The supernatant was decanted carefully and neutralized by the Freon/alamine method of Khym (14). The precipitated protein was measured by the Lowry et al. method (15).
Analysis
of phosphorylated
compounds.
Neutralized extract (50- 100 ~1) was placed on a 150 X 3-mm anion-exchange column connected to the automatic phosphate analyzer ( 16,17) and eluted according to the method described previously ( 12,18). The chromatogram in Fig. 1 illustrates the separation routinely obtained. The duplicate 20-nmol internal standards are also shown. The radioactivity in each peak was quantified by collecting the effluent from the calorimeter in I-min fractions in plastic minivials (0.5 ml per vial). About 5.5 ml of scintillation fluid (Beta-Phase) was added to each vial with vigorous mixing and all vials were capped tightly and counted in a Delta-300 scintillation counter (Searle Analytic). Purljication process. The nucleotides of the CP3” solution were already removed by the activated charcoal absorption. To remove the remaining inorganic radioactive phosphate, the calcium method of precipitation of Pi described by Seraydarian (19) with some modification was applied. The materials needed for the process are 3 M NaHC03 and the precipitant solution, which is a saturated solution of calcium hydroxide in 1% calcium chloride. This is made by preparing two solutions in an ice bath, one of equal weights of sodium hydroxide and water and the other of equal weights of calcium chloride (anhydrous) and
376
SAVABI.
GEIGER,
FIG. I. Chromatogram illustrating separation of the phosphorylated compounds of neutralized CP3’ extract, using 500 FCi 33P1 (A). and after dilution of radioactive P, with addition of phosphate buffer to 5 mM P, and precipitation of f’L by calcium method (B). The sample had been extracted with activated charcoal; therefore. there were no adenine nucleotides left. The duplicate 20-nmol standards used for calculating the amount of phosphate in each peak are also shown. 0 - - - 0, Radioactivity (wm).
water. Mix in an ice bath equal parts of the two solutions, causing a massive precipitation of calcium hydroxide. This precipitate is filtered through a Buchner funnel using two sheets of Whatman No. 2 filter paper. The paste on the funnel is washed twice with water and twice with 1% calcium chloride solution. About a cubic centimeter of the freshly prepared paste is mixed vigorously in a test tube with 10 ml of 1% calcium chloride and centrifuged at 2000 rpm for 2 min. The supernatant, which may be slightly turbid, is stop-
AND
BESSMAN
pered to exclude CO- and is used as precipitating reagent. The neutralized CP3j solution, about 2.52.6 ml. was placed in a 10 ml test tube. The pH was adjusted to about 9 (phenolphthalein) with 1 N NaOH. A few hundred microliters of 50 mM phosphate buffer (pH 7.2) was added as carrier. Preliminary experiments showed that at least 1.5-2 times the molar ratio of Ca(OH)2 to phosphate was needed to precipitate more than 98% of the inorganic phosphate. An excess of Ca(OH)2 also precipitates adenine nucleotides, but we found this to be incomplete so we used the charcoal step for complete removal of adenine nucleotides. Since the saturated Ca(OH)2 in cold water is 25 InM, between 0.75 and 1 ml of precipitant solution, depending on the concentration of P,, was usually added to the crude CP33 solution (pH -9). and 10 to 20 ~1 of 3 M NaHC03 was then added. The procedures were carried out in an ice bath. After 5 min in ice, the fine precipitate of calcium phosphate was centrifuged down at 3000 rpm for 3 min. The supernatant was decanted very carefully and a small portion (usually 100 ~1) was placed on the AGMP- 1 resin of the phosphate analyzer for analysis. The final solution has a large volume, and also contains mannitol and salts. To remove these salts and decrease the volume, the 33Pcreatine phosphate solution (volume more than 3 ml), pH 8.5, was placed in a 3-ml syringe barrel containing 0.7 ml of AGMP-1 resin, prepared as described for analysis of phosphorylated compounds ( 12,18). The resin was then washed with alkaline (ammonium hydroxide) water (pH 8.5 to 9). The 33P-creatine phosphate was then eluted with 1.5 ml of 0.7 M HC104 solution in 3 X 0.5-ml steps. The entire yield of CP33 could be obtained in about 1.6-1.7 ml by pushing the plunger of the syringe down to the resin bed after the third addition of eluant. This solution was extracted by the Freon/alamine method of Khym (14) to remove the HC104 and then was neutralized. This solution 25 to 50 ~1 was used for the final analysis.
“P-CREATINE
RESUILTS
AND
PHOSPHATE
mM P, final concentration), 0.5 ml of Hz0 or “Pi solution, 5.2 mg of creatine, 7 ~1 of ADP solution (final 70 PM), 75 ~1 of 0.1 M cr-ketoglutarate, and 0.5 ml of mitochondrial suspension and wasincubated for 10 min at 29°C with vigorous shaking. In the presence of 2 mM phosphate, more than 95% of Pi was converted to CP. Using 500 &I, j3Pi gave slightly less (about 92%). If more radioactive “PI was used (0.8 mCi) about 86.1% of the P, was converted to CP. This might be due to radiation damage of mitochondria, which needsfurther investigation. The results are summarized in Table 2. The calcium method of precipitation of Pi removed more than 99.7% of the radioactive Pi. The two-step purification processremoved all detectable counts in adenine nucleotides. Table 3 shows a summary of the results of different purification processes.Figure I is a representative chromatogram from the au-
DISCUSSION
The resultsfrom several experiments on the synthesis of CP, as various conditions were applied to optimize the yield and minimize contaminatio’n, are shown in Table 1. It was found that the more mitochondrial protein used,the more CP synthesized. The maximum amount of CP synthesized in 10 min. under the best conditions, was about 0.6 pmol/mg of mitochondrial protein. For a given amount of mitochondria, an increasein concentration of phosphateIdid not increasethe total amount of synthesized CP. Increasing the concentration of ADP from 50 to 460 PM causeda slight linear decreasein the final yield of CP. Five to ten minutes of incubation was enough for maximum synthesis. Mitochondrial suspension (0.5 ml) containing 8-9 mg protein was usedfor the best results. The 1.582-ml reaction volume contained 0.5 ml of the cocktail (2
TABLE INCuBATIOr4
N heart I
2
3
4
5
CONDITIONS
FOR CREATINE
Mitochondrial protein (mg)
ADP (PM)
1 (CP) SYNTHESIS
PHOSPHATE
C (rnM)
BY RABBIT
Incubation time at 28°C
100 100 100
2
5
6.1 6.1
2 2
15 30
6.15 6.15 6.15
100 100 100
2
2
2 2
5
10
2 2 2
100 100 100
2 4 8
2.5 2.5
100
6.1
50
2.5 2.5
230
1.7
100 100
2.56
7.7 7.7
377
SYNTHESIS
460
100 100 AMP
Note. Polarographic studies were carried out to ascertain the quality respiratory control ratio. (For detail please see Materiak and Methods.)
HEART
MITOCHONDRIA
CP (mM) 1.867 1.857
Percentage conversion of P, to CP 93.35
1.656
92.85 82.8
1.373 1.871 1.889
68.65 93.55 94.45
10 10 10
0.694 0.683
34.1 17.08 8.5
10 10 10 10
0.798 0.762 0.718
39.9
0.682
34.1
10 10 10 10
0.591 0.798
14.78 19.95 85.75
of mitochondrial
0.680
3.43 3.636
preparations
38.1 35.9
90.9
in terms
of
378
SAVABI, TABLE
SUMMARY SYNTHESIS
Mitochondrial protein 9.86 9.95 9.82
GEIGER,
2
OF RESULTS
ON CREATINE
BY RABBIT INHIBITION
HEART MIT~CHONDRIA BY EXCESS “P,
“P I (mCi)
CP (pm00
0 0.5 0.8
2.99 2.91 2.73
PHOSPHATE
Percentage of P, converted to CP 94.33 91.7 86.0
Note. The 1.582-ml reaction mixture contained 0.5 ml mitochondrial suspension, 0.5 ml HZ0 + 33Pi, 0.5 ml of the cocktail (final concentration of Pi was 2 mM), 7 ~1 of ADP solution (final 70 PM), 75 pl of n-ketoglutarate solution (final 5 mM), and 5.2 mg creatine (final 25 mM). For details, please see text.
tomated phosphate analyzer showing Pi and 33Pi-creatinephosphate peaks before and after removal of 33Pi. The calcium method per se did not remove the CP. The activated charcoal extraction, however, did remove 5 to 8% of the CP3’. The overall loss due to purification is about 10%and thus, the final yield is above 75% (for 0.8 mCi 33Pi)and 80% (for 0.5 mCi 33Pi)on the basisof the original Pi. The 10s~ of 33P-creatinephosphate used in chromatography for the analysis has not been included, but usually lessthan 2% of total radioactivity was used for the analysis.
AND
The present work describesa simple. rapid, high-yield method to synthesize 33P-creatine phosphate of high purity by using heart mitochondria asthe enzyme source. This method can be very useful for the synthesis of radioactive creatine phosphate especially in the case of 33Pi,since [T-~~P]ATP is not commercially available. In view of the fact that only mitochondria from heart, skeletal muscle, and brain contain significant amounts of bound CPK (20) mitochondria from only these tissuescan be usedfor this method. Experimental work in this laboratory hasshown that creatine phosphate synthesis does occur in mitochondrial preparations from all of the above tissues. but we have only used rabbit heart mitochondria for the present method.
TABLE A SUMMARY
OF THE RESULTS
BESSMAN
REFERENCES I. Bessman, S. P., and Geiger, P. J. (198 I ) Science 211, 448-452. 2. Shih. Yen-Shiang. and Whitesides, G. M. (1977) J
Org. Chem. 42,4165-4166. 3. Mokvosch.
L. D., Caravaca,
J.. and Grisolia,
S. ( 1960)
Biochim. Biophys. Actu 31, 442-447. 4. Metzenberg, R. L.. Marshall. M.. Cohen, Miller, W. G. (1959) J. Biol. Chem. 1537. 5. Lowe, G.. and Sproat, B. S. (1979) J.
P. P., and
234, 1534Chem. Sot.
Perkin Trans. I, 1622- 1630. 3
ON PURIFICATION
PROWESSES
OF SYNTHESIZED
CP33
AN
CP pi 33P (mCi)
pm01
I
0.5 0.8
-
-
11
0.5 0.8
2.57 2.45
III
0.5 0.8
2.50 2.39
Total
cpm
SA &i/pm01
pm01
Total wm
Loss” WI
1.06 x lo9 1.76 X IO9
0.11 0.11
-
-
0.24 0.44
0.08 x IO9 0.24 X IO9
NDb ND
2316 4549
7.96 5.30
0.26 0.19
0.07 X 10’ 0.15 X 10’
ND ND
ND ND
2.7 2.4
firno1
Total
-
3.17 3.17
0.86 x lo9 1.35 x lo9
387 608
0.83 x lo9 1.33 x lo9
373 599
Note. I: Content of the reaction mixture at zero time. II: After III: Final purified CP33 solution. AN is total adenine nucleotides. ’ Loss of synthesized CP33 by the purification step. b Not detectable.
cpm
CP-” formation
and activated
charcoal
extraction.
“P-CREATINE 6. Cohen. M., and Hu. A. (1980) J. .4mer. 102(3). 913-916. 7. Yagi. K., and Noda. L. (1960) Bioctzirn.
PHOSPHATE
Chem. Sot Bwptz?:T.
Acta 43, 249-259. Pediatr. 56, 191-203. 9. Bessman, S. P., and Fonyo, A. (1966) Biochem. Biophj:v. Rex Commun. 22, 597-602. 8. Bessman.
S. I’. (1960)
J.
10. Saks. V. A.. Chernousova. G. B., Gukovsky. Smirnov. V. N., and Chazov. E. 1. (1975) RiwtleFn. 9, 273-290. 11. Saks, V. A.. Lipina. zov, E. I. (1976) 41.
N. V., Smirnov,
D. E., Eur. .I.
V. N.. and Cha-
Arch. Biochem. Biophja. 173, 34-
12. Yang, W. C. T.. Geiger, P. J.. Bessman, S. P.. and Borrebaek, B. (1977) Biochem. Biophys. Res. ~‘w~7Fmuz. ‘76, 882-887.
SYNTHESIS
379
B. J.. and Bessman, S. P. (1973) .4rch. Enwron. Heulrh 27. 112-l 15. 14. Khym, J. X. (1975) Clin. Chem. 21, 1245-1252.
13. Dyce,
IS. Lowry. 0. H., Rosebrough. A. J.. Farr. R. L.. and Randall. R. J. (1951) J. Biot. Chem. 193, 265275. 16. Bessman, S. P. (1974) .4na/. Biochem. 59. 524-532. 17. Bessman. S. P.. Geiger, P. J., Lu, T. C.. and McCabe, E. R. B. (1974) Anal. B&hem 59, 533-546. 18. Geiger, P. J., Ahn. S., and Bessman, S. P. (1980) in Methods in Carbohydrate Chemistry (Whistler. R.. ed.). pp. 21-32. Academic Press, New York. 19. Seraydarian. K., Mommaerts. W. F. H. M., Wallner. A., and Guillory. R. J. ( 1961) J. Bid Chem 236, 207 I-2075. 20. Klingenberg, M., and Schollmeyer. P. (1960) Biochem. %. 333, 335-340.
BIOCHEMISTRY
Three-Step
137, 374-379
Preparation and Purification of Phosphorus-33-Labeled Creatine Phosphate of High Specific Activity F. SAVABI,
lfniversit.v
(I 984)
of Southern
Ca@rnia
P. J. GEIGER,
School
r$ MedicGe, Received
AND S. P. BESSMAN 2025 Zonal.4venue,
August
Los Angeles,
Califbrnia
90033
5, 1983
Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P,) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P, is described. KEY WORDS: creatine phosphate: synthesis; inorganic phosphate removal: radioactive phosphate.
Radioautography studies of the creatine phosphate (CP)’ energy shuttle (1) required phosphorus-labeledcreatine phosphate of high purity to record the movements of the phosphate group during the contraction process. 33Pihas much too high energy (max 1710 kV) for electron micrographic studies, but it appears possible to use 33Pias a label because its energy (max 248 kV) is lessthan twice as high as 14C(max 156 kV). The design of the experiment required as little contamination as possible with labeled adenine nucleotides. The synthesis of radioactive creatine phosphate used in biochemical reactions has been carried out with purified creatine phosphokinase (EC 2.7.3.2) and a system for regenerating labeled ATP usually with a final yield of lessthan 25% based on radioactive phosphate (2). Phosphorus-labeled creatine phosphate hasalsobeen synthesized from y-labeled ATP, prepared with synthetic labeled carbamyl-P (3-6). The yield of ATP in this case, before several steps of purification, is about 75% on the basis of Pi. Yagi and Noda in ’ Abbreviations used: CP, creatine phosphate: creatine phosphokinase; CP 33. ‘jP-labeled creatine phate. 0003-2697184
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
CPK, phos-
374
1960 (7) used liver mitochondria for the synthesis of [T-~~P]ATP. The synthesized [y32P]ATP was then purified and used for the enzymatic synthesisof 32P-creatinephosphate. The final purified 32P-creatinephosphate still contained 5% Pi and the overall yield was 36% on the basis of 32Pi.We present here a highyield simplified procedure for direct mitochondrial synthesisof 33P,-creatinephosphate. Our method is based on the coupled reaction of mitochondrial oxidative phosphorylation and mitochondrial-bound creatine phosphokinase (CPK) (8- I I), using rabbit heart mitochondria, excesscreatine, inorganic 33Piphosphate, and 50-70 PM ADP. MATERIALS Male
New Zealand
AND white
METHODS rabbits,
6-7
lb.,
were obtained from ABC Rabbitry, Rosemead, California. Trizma base, creatine, nucleotides, dithiothreitol (DTT), and EDTA were purchased from Sigma Chemical, St. Louis, Missouri. Alamine(tri-n-octyl tertiaryamine) was purchased from McKerson Corporation, Minneapolis, Minnesota. FreonTP was obtained from Miller-Stephenson Company, Danbury. Connecticut. Beta-Phase
33P-CREATINE
PHOSPHATE
scintillation1 cocktail was purchased from WestChem Products, Inc., San Diego, California. Isotope 33P-H3P04 was obtained from New England Nuclear (Boston, Mass.). Activated charcoal was obtained from Matheson, Coleman and Bell, Cincinnati, Ohio.
Preparation
qf rabbit heart mitochondria.
Mitochondria were isolated from the ventricles of New Zealand albino rabbits according to the method of Yang et al. ( 1977) (12) with substitution of Tris-Cl for Tris-PO4 in all solutions. The homogenization medium contained 0.35 ‘M mannitol, 0.1 mM EDTA, and 10 mM T&-Cl (pH 7.3). Suspending medium contained. in addition to the above, 0.5% dialyzed bovine serum albumin. Polarographic studies were carried out on all preparations and only those having respiratory control ratios higher than 11 in the absence of Mg2+ were used.
Incubation conditions and protocol,for creatine phosphate synthesis. To optimize the conditions for the synthesis of creatine phosphate, several variables, including concentration of inorganic phosphate, ADP, mitochondrial protein, and different incubation times, were tested. The 1.582-ml (sum of additions) reaction mixture contained 0.5 ml “cocktail” [consisting of 0.543 M mannitol, 0.2 17 mM EDTA, 2 1.5’ mM Tris-Cl (pH 7.2) 3 1.7 mM KCI, 0.3 17 mM DTT, 6.34 mM MgCl?, and different concentrations of potassium phosphate buffer (pH 7.2)], 0.5 ml Hz0 or 33Pi solution, 0 to 0.3 ml suspending medium, 0.5 to 0.2 ml mitochondrial suspension. 7 ~1 of ADP solution, 75 ~1 of 0.1 M cu-ketoglutarate, and 5.2 mg of crystalline creatine. The final reaction medium contained 0.25 M mannitol, 10 mM KCl, 10 mM Tris-Cl (pH 7.2). 0.1 mM EDTA, 0.1 mM DTT, 2 mM MgC12, 25 mM creatine, 5 mM a-ketoglutarate, and various concentrations of either ADP, Pi. or mitochondrial protein. The creatine was added to 0.5 ml cocktail in about 15 X loo-mm plastic tube., 0.5 ml Hz0 plus or minus 3’Pi was then added. and the mixture was shaken until the creatine was dissolved. ADP and (Yketoglutarate were then added and the reaction
SYNTHESIS
375
was started by addition of mitochondrial suspension. The open test tube containing this mixture was then shaken at 3/s at 20°C. The reaction was stopped by addition of 1 ml of 1.4 M HC104 and centrifuged at 3000 rpm for 10 min. The supernatant solution was decanted carefully. At this point unlabeled ATP and ADP were added to the supernatant solution to final 3 and 2 mM concentrations, respectively, to dilute the radioactive nucleotides. About 55 mg activated charcoal was then added to the solution, which was centrifuged at 3000 r-pm for 5 min ( 13). The supernatant was decanted carefully and neutralized by the Freon/alamine method of Khym (14). The precipitated protein was measured by the Lowry et al. method (15).
Analysis
of phosphorylated
compounds.
Neutralized extract (50- 100 ~1) was placed on a 150 X 3-mm anion-exchange column connected to the automatic phosphate analyzer ( 16,17) and eluted according to the method described previously ( 12,18). The chromatogram in Fig. 1 illustrates the separation routinely obtained. The duplicate 20-nmol internal standards are also shown. The radioactivity in each peak was quantified by collecting the effluent from the calorimeter in I-min fractions in plastic minivials (0.5 ml per vial). About 5.5 ml of scintillation fluid (Beta-Phase) was added to each vial with vigorous mixing and all vials were capped tightly and counted in a Delta-300 scintillation counter (Searle Analytic). Purljication process. The nucleotides of the CP3” solution were already removed by the activated charcoal absorption. To remove the remaining inorganic radioactive phosphate, the calcium method of precipitation of Pi described by Seraydarian (19) with some modification was applied. The materials needed for the process are 3 M NaHC03 and the precipitant solution, which is a saturated solution of calcium hydroxide in 1% calcium chloride. This is made by preparing two solutions in an ice bath, one of equal weights of sodium hydroxide and water and the other of equal weights of calcium chloride (anhydrous) and
376
SAVABI.
GEIGER,
FIG. I. Chromatogram illustrating separation of the phosphorylated compounds of neutralized CP3’ extract, using 500 FCi 33P1 (A). and after dilution of radioactive P, with addition of phosphate buffer to 5 mM P, and precipitation of f’L by calcium method (B). The sample had been extracted with activated charcoal; therefore. there were no adenine nucleotides left. The duplicate 20-nmol standards used for calculating the amount of phosphate in each peak are also shown. 0 - - - 0, Radioactivity (wm).
water. Mix in an ice bath equal parts of the two solutions, causing a massive precipitation of calcium hydroxide. This precipitate is filtered through a Buchner funnel using two sheets of Whatman No. 2 filter paper. The paste on the funnel is washed twice with water and twice with 1% calcium chloride solution. About a cubic centimeter of the freshly prepared paste is mixed vigorously in a test tube with 10 ml of 1% calcium chloride and centrifuged at 2000 rpm for 2 min. The supernatant, which may be slightly turbid, is stop-
AND
BESSMAN
pered to exclude CO- and is used as precipitating reagent. The neutralized CP3j solution, about 2.52.6 ml. was placed in a 10 ml test tube. The pH was adjusted to about 9 (phenolphthalein) with 1 N NaOH. A few hundred microliters of 50 mM phosphate buffer (pH 7.2) was added as carrier. Preliminary experiments showed that at least 1.5-2 times the molar ratio of Ca(OH)2 to phosphate was needed to precipitate more than 98% of the inorganic phosphate. An excess of Ca(OH)2 also precipitates adenine nucleotides, but we found this to be incomplete so we used the charcoal step for complete removal of adenine nucleotides. Since the saturated Ca(OH)2 in cold water is 25 InM, between 0.75 and 1 ml of precipitant solution, depending on the concentration of P,, was usually added to the crude CP33 solution (pH -9). and 10 to 20 ~1 of 3 M NaHC03 was then added. The procedures were carried out in an ice bath. After 5 min in ice, the fine precipitate of calcium phosphate was centrifuged down at 3000 rpm for 3 min. The supernatant was decanted very carefully and a small portion (usually 100 ~1) was placed on the AGMP- 1 resin of the phosphate analyzer for analysis. The final solution has a large volume, and also contains mannitol and salts. To remove these salts and decrease the volume, the 33Pcreatine phosphate solution (volume more than 3 ml), pH 8.5, was placed in a 3-ml syringe barrel containing 0.7 ml of AGMP-1 resin, prepared as described for analysis of phosphorylated compounds ( 12,18). The resin was then washed with alkaline (ammonium hydroxide) water (pH 8.5 to 9). The 33P-creatine phosphate was then eluted with 1.5 ml of 0.7 M HC104 solution in 3 X 0.5-ml steps. The entire yield of CP33 could be obtained in about 1.6-1.7 ml by pushing the plunger of the syringe down to the resin bed after the third addition of eluant. This solution was extracted by the Freon/alamine method of Khym (14) to remove the HC104 and then was neutralized. This solution 25 to 50 ~1 was used for the final analysis.
“P-CREATINE
RESUILTS
AND
PHOSPHATE
mM P, final concentration), 0.5 ml of Hz0 or “Pi solution, 5.2 mg of creatine, 7 ~1 of ADP solution (final 70 PM), 75 ~1 of 0.1 M cr-ketoglutarate, and 0.5 ml of mitochondrial suspension and wasincubated for 10 min at 29°C with vigorous shaking. In the presence of 2 mM phosphate, more than 95% of Pi was converted to CP. Using 500 &I, j3Pi gave slightly less (about 92%). If more radioactive “PI was used (0.8 mCi) about 86.1% of the P, was converted to CP. This might be due to radiation damage of mitochondria, which needsfurther investigation. The results are summarized in Table 2. The calcium method of precipitation of Pi removed more than 99.7% of the radioactive Pi. The two-step purification processremoved all detectable counts in adenine nucleotides. Table 3 shows a summary of the results of different purification processes.Figure I is a representative chromatogram from the au-
DISCUSSION
The resultsfrom several experiments on the synthesis of CP, as various conditions were applied to optimize the yield and minimize contaminatio’n, are shown in Table 1. It was found that the more mitochondrial protein used,the more CP synthesized. The maximum amount of CP synthesized in 10 min. under the best conditions, was about 0.6 pmol/mg of mitochondrial protein. For a given amount of mitochondria, an increasein concentration of phosphateIdid not increasethe total amount of synthesized CP. Increasing the concentration of ADP from 50 to 460 PM causeda slight linear decreasein the final yield of CP. Five to ten minutes of incubation was enough for maximum synthesis. Mitochondrial suspension (0.5 ml) containing 8-9 mg protein was usedfor the best results. The 1.582-ml reaction volume contained 0.5 ml of the cocktail (2
TABLE INCuBATIOr4
N heart I
2
3
4
5
CONDITIONS
FOR CREATINE
Mitochondrial protein (mg)
ADP (PM)
1 (CP) SYNTHESIS
PHOSPHATE
C (rnM)
BY RABBIT
Incubation time at 28°C
100 100 100
2
5
6.1 6.1
2 2
15 30
6.15 6.15 6.15
100 100 100
2
2
2 2
5
10
2 2 2
100 100 100
2 4 8
2.5 2.5
100
6.1
50
2.5 2.5
230
1.7
100 100
2.56
7.7 7.7
377
SYNTHESIS
460
100 100 AMP
Note. Polarographic studies were carried out to ascertain the quality respiratory control ratio. (For detail please see Materiak and Methods.)
HEART
MITOCHONDRIA
CP (mM) 1.867 1.857
Percentage conversion of P, to CP 93.35
1.656
92.85 82.8
1.373 1.871 1.889
68.65 93.55 94.45
10 10 10
0.694 0.683
34.1 17.08 8.5
10 10 10 10
0.798 0.762 0.718
39.9
0.682
34.1
10 10 10 10
0.591 0.798
14.78 19.95 85.75
of mitochondrial
0.680
3.43 3.636
preparations
38.1 35.9
90.9
in terms
of
378
SAVABI, TABLE
SUMMARY SYNTHESIS
Mitochondrial protein 9.86 9.95 9.82
GEIGER,
2
OF RESULTS
ON CREATINE
BY RABBIT INHIBITION
HEART MIT~CHONDRIA BY EXCESS “P,
“P I (mCi)
CP (pm00
0 0.5 0.8
2.99 2.91 2.73
PHOSPHATE
Percentage of P, converted to CP 94.33 91.7 86.0
Note. The 1.582-ml reaction mixture contained 0.5 ml mitochondrial suspension, 0.5 ml HZ0 + 33Pi, 0.5 ml of the cocktail (final concentration of Pi was 2 mM), 7 ~1 of ADP solution (final 70 PM), 75 pl of n-ketoglutarate solution (final 5 mM), and 5.2 mg creatine (final 25 mM). For details, please see text.
tomated phosphate analyzer showing Pi and 33Pi-creatinephosphate peaks before and after removal of 33Pi. The calcium method per se did not remove the CP. The activated charcoal extraction, however, did remove 5 to 8% of the CP3’. The overall loss due to purification is about 10%and thus, the final yield is above 75% (for 0.8 mCi 33Pi)and 80% (for 0.5 mCi 33Pi)on the basisof the original Pi. The 10s~ of 33P-creatinephosphate used in chromatography for the analysis has not been included, but usually lessthan 2% of total radioactivity was used for the analysis.
AND
The present work describesa simple. rapid, high-yield method to synthesize 33P-creatine phosphate of high purity by using heart mitochondria asthe enzyme source. This method can be very useful for the synthesis of radioactive creatine phosphate especially in the case of 33Pi,since [T-~~P]ATP is not commercially available. In view of the fact that only mitochondria from heart, skeletal muscle, and brain contain significant amounts of bound CPK (20) mitochondria from only these tissuescan be usedfor this method. Experimental work in this laboratory hasshown that creatine phosphate synthesis does occur in mitochondrial preparations from all of the above tissues. but we have only used rabbit heart mitochondria for the present method.
TABLE A SUMMARY
OF THE RESULTS
BESSMAN
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Org. Chem. 42,4165-4166. 3. Mokvosch.
L. D., Caravaca,
J.. and Grisolia,
S. ( 1960)
Biochim. Biophys. Actu 31, 442-447. 4. Metzenberg, R. L.. Marshall. M.. Cohen, Miller, W. G. (1959) J. Biol. Chem. 1537. 5. Lowe, G.. and Sproat, B. S. (1979) J.
P. P., and
234, 1534Chem. Sot.
Perkin Trans. I, 1622- 1630. 3
ON PURIFICATION
PROWESSES
OF SYNTHESIZED
CP33
AN
CP pi 33P (mCi)
pm01
I
0.5 0.8
-
-
11
0.5 0.8
2.57 2.45
III
0.5 0.8
2.50 2.39
Total
cpm
SA &i/pm01
pm01
Total wm
Loss” WI
1.06 x lo9 1.76 X IO9
0.11 0.11
-
-
0.24 0.44
0.08 x IO9 0.24 X IO9
NDb ND
2316 4549
7.96 5.30
0.26 0.19
0.07 X 10’ 0.15 X 10’
ND ND
ND ND
2.7 2.4
firno1
Total
-
3.17 3.17
0.86 x lo9 1.35 x lo9
387 608
0.83 x lo9 1.33 x lo9
373 599
Note. I: Content of the reaction mixture at zero time. II: After III: Final purified CP33 solution. AN is total adenine nucleotides. ’ Loss of synthesized CP33 by the purification step. b Not detectable.
cpm
CP-” formation
and activated
charcoal
extraction.
“P-CREATINE 6. Cohen. M., and Hu. A. (1980) J. .4mer. 102(3). 913-916. 7. Yagi. K., and Noda. L. (1960) Bioctzirn.
PHOSPHATE
Chem. Sot Bwptz?:T.
Acta 43, 249-259. Pediatr. 56, 191-203. 9. Bessman, S. P., and Fonyo, A. (1966) Biochem. Biophj:v. Rex Commun. 22, 597-602. 8. Bessman.
S. I’. (1960)
J.
10. Saks. V. A.. Chernousova. G. B., Gukovsky. Smirnov. V. N., and Chazov. E. 1. (1975) RiwtleFn. 9, 273-290. 11. Saks, V. A.. Lipina. zov, E. I. (1976) 41.
N. V., Smirnov,
D. E., Eur. .I.
V. N.. and Cha-
Arch. Biochem. Biophja. 173, 34-
12. Yang, W. C. T.. Geiger, P. J.. Bessman, S. P.. and Borrebaek, B. (1977) Biochem. Biophys. Res. ~‘w~7Fmuz. ‘76, 882-887.
SYNTHESIS
379
B. J.. and Bessman, S. P. (1973) .4rch. Enwron. Heulrh 27. 112-l 15. 14. Khym, J. X. (1975) Clin. Chem. 21, 1245-1252.
13. Dyce,
IS. Lowry. 0. H., Rosebrough. A. J.. Farr. R. L.. and Randall. R. J. (1951) J. Biot. Chem. 193, 265275. 16. Bessman, S. P. (1974) .4na/. Biochem. 59. 524-532. 17. Bessman. S. P.. Geiger, P. J., Lu, T. C.. and McCabe, E. R. B. (1974) Anal. B&hem 59, 533-546. 18. Geiger, P. J., Ahn. S., and Bessman, S. P. (1980) in Methods in Carbohydrate Chemistry (Whistler. R.. ed.). pp. 21-32. Academic Press, New York. 19. Seraydarian. K., Mommaerts. W. F. H. M., Wallner. A., and Guillory. R. J. ( 1961) J. Bid Chem 236, 207 I-2075. 20. Klingenberg, M., and Schollmeyer. P. (1960) Biochem. %. 333, 335-340.