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Separation of 32P-labeled inorganic phosphate from rat liver homogenates
ANALYTICAL
BIOCHEMISTRY
Separation
77, 548-551 (1977)
of 32P-Labeled Inorganic Phosphate from Rat Liver Homogenates
A method has been developed for the separation of radioactive inorganic phosphate from rat liver homogenates by a combination of ion-exchange and precipitation chromatography. The method has been applied to normal rat liver.
In order to determine accurately the specific activity of rat liver phosphate after injection of a tracer dose of 32P-labeled inorganic phosphate, it is necessary to separate the inorganic phosphate from the other compounds which have also become labeled over the time course of the experiment. Among the other compounds which become labeled within 30 min are the nucleotides (especially adenine) and sugar phosphates (1). In the method described here, two ion-exchange resins are utilized to isolate the inorganic phosphate (orthophosphate) from deproteinized rat liver homogenate. The nucleotides are first removed by an anionexchange column (strong-base resin in chloride form). The inorganic phosphate is removed from the eluant by passage through a cationexchange resin which is in the lanthanum (La+3) form. The phosphate is retained, probably by forming a precipitate of extremely insoluble lanthanum phosphate (LaPO,) within the resin particles; the sugar phosphates pass through. Both the nucleotides and the inorganic phosphate can be eluted from their respective resins with 1 N hydrochloric acid. Thus isolated, the inorganic phosphate can then be counted and quantitated by conventional techniques so that its specific activity can be calculated. In this paper, the method and its application to the analysis of real and simulated rat liver homogenates are described. METHODS Apparatus. Ion-exchange columns were Bio-Rad EconoColumns, 0.7cm internal diameter x lo-cm length, filled to a height of 4 cm with either Bio-Rad AG l-X8 (200-400 mesh) anion-exchange resin or Bio-Rad AG 5OW-X8 (200-400 mesh) cation-exchange resin (Bio-Rad Laboratories, Richmond, Calif.). Reagents. A standard solution was prepared which contained 0.42 mM 32P-labeled inorganic phosphate (1 &i/ml), 0.14 mM ATP, 0.14 mM ADP, 0.14 mM AMP, 0.07 mM [14C]glucose l-phosphate (0.5 $Zi/ml), and 0.07 mM [14C]glucose 6-phosphate (0.5 &i/ml), and the solution was adjusted to pH 7. This standard was a model of a liver homogenate (2). Procedure. The anion-exchange columns were each washed with 4 ml of 1 N HCl and then with water until neutral. The cation-exchange 548 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
549
SHORT COMMUNICATIONS TABLE RECOVERY
Standard solution Range Mean t SEM(N) Median
OF 32P-~~~~~~~
1 INORGANIC
PHOSPHATE
Nucleotide removed (%I
Phosphate recovery (%I
Sugar phosphate contaminant (%I
95.1-106.9 101 f 0.9 (13) 100.5
94.2- 105.2 98.4 k 1.1 (9) 98.0
o-14.1 5.0 2 1.4 (13) 4.0
Phosphate recovery Homogenate Range Mean h SEM(N) Median
93.2- 104.8 100.8 +- 0.8 (19) 101.8
columns were treated in the same way, then washed twice with lo-ml portions of 0.1 M lanthanum chloride (LaCl,) followed by water until the eluate was free of chloride. The outlet of an anion-exchange column was connected to the inlet of a cation-exchange column by means of a three-way stopcock. A l-ml aliquot of the standard was applied to the anion-exchange (upper) column and followed with a wash of 5 ml of water. The column was eluted with two lo-ml portions of 0.025 M NH&l at a flow rate of 0.5 ml/min and then connected to a reservoir and eluted more rapidly with an additional 100 ml of 0.025 M NH&l. At the completion of elution, the columns were separated and each was eluted with two lo-ml portions of 1 N HCl. Nucleotides were determined by their ultraviolet (260 nm) absorbance, and [32P]phosphate and the [14C]glucose phosphates were counted in separate channels with a liquid scintillation spectrometer. To determine recovery of phosphate from rat liver homogenates, 1 g of liver was homogenized in 19 ml of ice-cold water. Approximately 1 &i/ml of 32P-labeled inorganic phosphate was added to the homogenate. Ten milliliters of 0.6 N HC104 were added and mixed, and the protein was allowed to settle in an ice bath for 10 min. The protein was centrifuged down, and the supernatant was neutralized with 5 N K&O,. KC104 was allowed to precipitate in the cold for 30 min and centrifuged at 90008 for 10 min. A 1S-ml aliquot of neutralized homogenate was applied to the anion-exchange (upper) column, and 4.5 ml of water were added. The column was then eluted with 0.025 M NH,CI as above. Phosphate was estimated as the malachite green complex with phosphomolybdate (3). Five normal Sprague-Dawley male rats (Bio-Lab, White Bear Lake, Minn.) were injected intraperitoneally with 0.207 mCi of 32P-labeled inorganic phosphate in 0.01 M phosphate buffer, pH 7.4,
550
SHORT
COMMUNICATIONS TABLE
FRACTIONATION
OF LABELED Fraction
Inorganic
phosphate
+- SEM
PHOSPHATE
FROM
RAT LIVER
(% of total 32P) Nucleotide
33.7 f 2.9 (5) a Mean
2
47.9 -I- 3.7 (5)
Glucose
metabolites
22.6 -+ 2.3 (5)
(N)
per 250 g of body weight. After 30 min, the rats were decapitated, of liver was processed as above. RESULTS
and 1 g
AND DISCUSSION
The results of the application of this method to fractionation of the standard solution are shown in Table 1. The nucleotides were successfully removed by the anion-exchange column, and 98.4% of the phosphate was retained by the lanthanum column. However, 5% of the glucose l-phosphate and glucose Bphosphate was retained by the lanthanum column. In addition, all of the added phosphate was recovered from a rat liver homogenate. The distribution of labeled phosphate after 30 min in normal rat liver was 34% as inorganic phosphate, 48% in the nucleotide fraction, and 23% in the glucose-metabolite fraction (Table 2). Thus, contamination of the phosphate by 5% of the glucose metabolites would result in an error of estimation of about 3%. For the determination of the specific activity of radioactive phosphate, both the amount of phosphate and the radioactivity have to be assayed. Of the many methods available for the estimation of phosphate, that of Fiske and SubbaRow is one of the simplest and most widely used (4). However, in this procedure, the extralabile phosphate compounds, i.e., phosphocreatine, 1,3-diphosphoglycerate, ribose l-phosphate, deoxyribose l-phosphate, and the arylphosphates, are hydrolyzed by the acid (pH 0.65), in some cases catalyzed by molybdate, and assayed as phosphate. In addition, significant portions of ATP and phosphoenolpyruvate are hydrolyzed to phosphate over the course of the reaction (4). An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds using the catalyst polyvinylpyrrolidone has recently been described (5). This method appears to be the method of choice for the analytical determination of phosphate in situations where it has adequate sensitivity. In our hands, it is one-half as sensitive as the Fiske-SubbaRow procedure and one-tenth as sensitive as the Itaya-Ui (3) procedure. It is convenient to extract the phosphomolybdate complex with isobutanol (6,7) so that the same sample can be assayed for phosphate
551
SHORT COMMUNICATIONS
and radioactivity. However, as pointed out by Hagihara and Lardy (8), separation by this method is far from perfection, and the extralabile phosphates are still hydrolyzed due to low pH. It would be ideal for this purpose if the phosphomolybdate complex in the catalyst method (5) could be separated. However, we have been unable to extract the colored complex into organic solvents (chloroform, butanol, butylacetate) even after acidifying the solution, probably due to the presence of the catalyst polyvinylpyrrolidone. Therefore, for the determination of the specific activity of radioactive phosphate, the phosphate will have to be separated by another method and the radioactivity determined. In the method described in this paper, phosphate is first separated from the other labeled phosphate compounds by precipitation as lanthanum phosphate within the resin particles. After elution of the phosphate, the radioactivity is determined in one aliquot, and the phosphate is measured in another aliquot by the Itaya-Ui procedure (3). We have used this method to determine the specific activity of rat liver phosphate after administration of 32P-labeled inorganic phosphate. ACKNOWLEDGMENTS The excellent acknowledged.
technical
assistance of Mary Brunfiel
and Kay Draves is hereby
REFERENCES 1. Feige, B., Gimmler, H., Jeschke, W. D., and Simonis, W. (1969) J. Chromatogr. 41, 80-W. 2. Long, C. (1961) The Biochemists Handbook, p. 681, van Nostrand, New York. 3. Itaya, K., and Ui, M. (1966) Clin. Chim. Acta 14, 361-366. 4. Leloir, L. F., and Cardini, C. E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 843-844, Academic Press, New York. 5. Ohnishi, T., Gall, R. S., and Mayer, M. L. (1975) Anal. Biochem. 69, 261-267. 6. Berenblum, I., and Chain, E. (1938) Biochem. J. 32, 295-302. 7. Weil-Malherbe, H., and Green, R. H. (195l)Biochem. J. 49,286-292. 8. Hagihara, B., and Lardy, H. A. (1960) J. Bio/. &em. 235, 889-894. ROBERT F. DERR’ RODNEY OLSEN~ LESLIE ZIEVE Department Minneapolis University Minneapolis, Received
of Medicine Veterans Hospital of Minnesota Minnesota 55417 May 26, 1976; accepted
September
28, 1976
1 Author to whom correspondence should be directed. 2 Present address: Chemistry Department, Hamline University, 55104.
St. Paul, Minnesota
BIOCHEMISTRY
Separation
77, 548-551 (1977)
of 32P-Labeled Inorganic Phosphate from Rat Liver Homogenates
A method has been developed for the separation of radioactive inorganic phosphate from rat liver homogenates by a combination of ion-exchange and precipitation chromatography. The method has been applied to normal rat liver.
In order to determine accurately the specific activity of rat liver phosphate after injection of a tracer dose of 32P-labeled inorganic phosphate, it is necessary to separate the inorganic phosphate from the other compounds which have also become labeled over the time course of the experiment. Among the other compounds which become labeled within 30 min are the nucleotides (especially adenine) and sugar phosphates (1). In the method described here, two ion-exchange resins are utilized to isolate the inorganic phosphate (orthophosphate) from deproteinized rat liver homogenate. The nucleotides are first removed by an anionexchange column (strong-base resin in chloride form). The inorganic phosphate is removed from the eluant by passage through a cationexchange resin which is in the lanthanum (La+3) form. The phosphate is retained, probably by forming a precipitate of extremely insoluble lanthanum phosphate (LaPO,) within the resin particles; the sugar phosphates pass through. Both the nucleotides and the inorganic phosphate can be eluted from their respective resins with 1 N hydrochloric acid. Thus isolated, the inorganic phosphate can then be counted and quantitated by conventional techniques so that its specific activity can be calculated. In this paper, the method and its application to the analysis of real and simulated rat liver homogenates are described. METHODS Apparatus. Ion-exchange columns were Bio-Rad EconoColumns, 0.7cm internal diameter x lo-cm length, filled to a height of 4 cm with either Bio-Rad AG l-X8 (200-400 mesh) anion-exchange resin or Bio-Rad AG 5OW-X8 (200-400 mesh) cation-exchange resin (Bio-Rad Laboratories, Richmond, Calif.). Reagents. A standard solution was prepared which contained 0.42 mM 32P-labeled inorganic phosphate (1 &i/ml), 0.14 mM ATP, 0.14 mM ADP, 0.14 mM AMP, 0.07 mM [14C]glucose l-phosphate (0.5 $Zi/ml), and 0.07 mM [14C]glucose 6-phosphate (0.5 &i/ml), and the solution was adjusted to pH 7. This standard was a model of a liver homogenate (2). Procedure. The anion-exchange columns were each washed with 4 ml of 1 N HCl and then with water until neutral. The cation-exchange 548 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0003.2697
549
SHORT COMMUNICATIONS TABLE RECOVERY
Standard solution Range Mean t SEM(N) Median
OF 32P-~~~~~~~
1 INORGANIC
PHOSPHATE
Nucleotide removed (%I
Phosphate recovery (%I
Sugar phosphate contaminant (%I
95.1-106.9 101 f 0.9 (13) 100.5
94.2- 105.2 98.4 k 1.1 (9) 98.0
o-14.1 5.0 2 1.4 (13) 4.0
Phosphate recovery Homogenate Range Mean h SEM(N) Median
93.2- 104.8 100.8 +- 0.8 (19) 101.8
columns were treated in the same way, then washed twice with lo-ml portions of 0.1 M lanthanum chloride (LaCl,) followed by water until the eluate was free of chloride. The outlet of an anion-exchange column was connected to the inlet of a cation-exchange column by means of a three-way stopcock. A l-ml aliquot of the standard was applied to the anion-exchange (upper) column and followed with a wash of 5 ml of water. The column was eluted with two lo-ml portions of 0.025 M NH&l at a flow rate of 0.5 ml/min and then connected to a reservoir and eluted more rapidly with an additional 100 ml of 0.025 M NH&l. At the completion of elution, the columns were separated and each was eluted with two lo-ml portions of 1 N HCl. Nucleotides were determined by their ultraviolet (260 nm) absorbance, and [32P]phosphate and the [14C]glucose phosphates were counted in separate channels with a liquid scintillation spectrometer. To determine recovery of phosphate from rat liver homogenates, 1 g of liver was homogenized in 19 ml of ice-cold water. Approximately 1 &i/ml of 32P-labeled inorganic phosphate was added to the homogenate. Ten milliliters of 0.6 N HC104 were added and mixed, and the protein was allowed to settle in an ice bath for 10 min. The protein was centrifuged down, and the supernatant was neutralized with 5 N K&O,. KC104 was allowed to precipitate in the cold for 30 min and centrifuged at 90008 for 10 min. A 1S-ml aliquot of neutralized homogenate was applied to the anion-exchange (upper) column, and 4.5 ml of water were added. The column was then eluted with 0.025 M NH,CI as above. Phosphate was estimated as the malachite green complex with phosphomolybdate (3). Five normal Sprague-Dawley male rats (Bio-Lab, White Bear Lake, Minn.) were injected intraperitoneally with 0.207 mCi of 32P-labeled inorganic phosphate in 0.01 M phosphate buffer, pH 7.4,
550
SHORT
COMMUNICATIONS TABLE
FRACTIONATION
OF LABELED Fraction
Inorganic
phosphate
+- SEM
PHOSPHATE
FROM
RAT LIVER
(% of total 32P) Nucleotide
33.7 f 2.9 (5) a Mean
2
47.9 -I- 3.7 (5)
Glucose
metabolites
22.6 -+ 2.3 (5)
(N)
per 250 g of body weight. After 30 min, the rats were decapitated, of liver was processed as above. RESULTS
and 1 g
AND DISCUSSION
The results of the application of this method to fractionation of the standard solution are shown in Table 1. The nucleotides were successfully removed by the anion-exchange column, and 98.4% of the phosphate was retained by the lanthanum column. However, 5% of the glucose l-phosphate and glucose Bphosphate was retained by the lanthanum column. In addition, all of the added phosphate was recovered from a rat liver homogenate. The distribution of labeled phosphate after 30 min in normal rat liver was 34% as inorganic phosphate, 48% in the nucleotide fraction, and 23% in the glucose-metabolite fraction (Table 2). Thus, contamination of the phosphate by 5% of the glucose metabolites would result in an error of estimation of about 3%. For the determination of the specific activity of radioactive phosphate, both the amount of phosphate and the radioactivity have to be assayed. Of the many methods available for the estimation of phosphate, that of Fiske and SubbaRow is one of the simplest and most widely used (4). However, in this procedure, the extralabile phosphate compounds, i.e., phosphocreatine, 1,3-diphosphoglycerate, ribose l-phosphate, deoxyribose l-phosphate, and the arylphosphates, are hydrolyzed by the acid (pH 0.65), in some cases catalyzed by molybdate, and assayed as phosphate. In addition, significant portions of ATP and phosphoenolpyruvate are hydrolyzed to phosphate over the course of the reaction (4). An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds using the catalyst polyvinylpyrrolidone has recently been described (5). This method appears to be the method of choice for the analytical determination of phosphate in situations where it has adequate sensitivity. In our hands, it is one-half as sensitive as the Fiske-SubbaRow procedure and one-tenth as sensitive as the Itaya-Ui (3) procedure. It is convenient to extract the phosphomolybdate complex with isobutanol (6,7) so that the same sample can be assayed for phosphate
551
SHORT COMMUNICATIONS
and radioactivity. However, as pointed out by Hagihara and Lardy (8), separation by this method is far from perfection, and the extralabile phosphates are still hydrolyzed due to low pH. It would be ideal for this purpose if the phosphomolybdate complex in the catalyst method (5) could be separated. However, we have been unable to extract the colored complex into organic solvents (chloroform, butanol, butylacetate) even after acidifying the solution, probably due to the presence of the catalyst polyvinylpyrrolidone. Therefore, for the determination of the specific activity of radioactive phosphate, the phosphate will have to be separated by another method and the radioactivity determined. In the method described in this paper, phosphate is first separated from the other labeled phosphate compounds by precipitation as lanthanum phosphate within the resin particles. After elution of the phosphate, the radioactivity is determined in one aliquot, and the phosphate is measured in another aliquot by the Itaya-Ui procedure (3). We have used this method to determine the specific activity of rat liver phosphate after administration of 32P-labeled inorganic phosphate. ACKNOWLEDGMENTS The excellent acknowledged.
technical
assistance of Mary Brunfiel
and Kay Draves is hereby
REFERENCES 1. Feige, B., Gimmler, H., Jeschke, W. D., and Simonis, W. (1969) J. Chromatogr. 41, 80-W. 2. Long, C. (1961) The Biochemists Handbook, p. 681, van Nostrand, New York. 3. Itaya, K., and Ui, M. (1966) Clin. Chim. Acta 14, 361-366. 4. Leloir, L. F., and Cardini, C. E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 843-844, Academic Press, New York. 5. Ohnishi, T., Gall, R. S., and Mayer, M. L. (1975) Anal. Biochem. 69, 261-267. 6. Berenblum, I., and Chain, E. (1938) Biochem. J. 32, 295-302. 7. Weil-Malherbe, H., and Green, R. H. (195l)Biochem. J. 49,286-292. 8. Hagihara, B., and Lardy, H. A. (1960) J. Bio/. &em. 235, 889-894. ROBERT F. DERR’ RODNEY OLSEN~ LESLIE ZIEVE Department Minneapolis University Minneapolis, Received
of Medicine Veterans Hospital of Minnesota Minnesota 55417 May 26, 1976; accepted
September
28, 1976
1 Author to whom correspondence should be directed. 2 Present address: Chemistry Department, Hamline University, 55104.
St. Paul, Minnesota