- Email: [email protected]
[32] Preparation and assay of phosphorylating submitochondrial particles
[32]
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
265
centrifugation, the enzyme is recovered without loss in the supernatant fluid, whereas 70% of the protein is discarded in the precipitate. Properties Enzyme fractions stored for weeks at --13 ° , often with repeated thawing and refreezing, show no signs of deterioration. Fractions 2 and 3, retested after 6 to 8 weeks, show no detectable loss in activity. The optimum pH of the reaction, determined in Tris buffers in the presence of a severalfold excess of acetokinase, is found to be near 7.2. The rates at pH 6.2, 6.7, 7.5, and 8.2 have been found to be 45, 75, 83, and 73%, respectively, of the rate at pH 7.2. At pH 7.2 the rate in phosphate buffer is about 25~6 slower than in Tris buffer. Ammonium sulfate stimulates the reaction rate of enzyme fraction 6; the rate in the presence of 0.04 M ammonium sulfate appears maximal and almost three times the rate in its absence. When the enzyme solubilized by ammonium sulfate was studied, the addition of ammonium sulfate did not stimulate the rate, although the concentration of the salt was only 0.002 M. Fluoride (5 X 10-* M) completely inhibits the enzyme; 2,4-dinitrophenol (3 X 10-4 M) does not inhibit. Magnesium is essential to the reaction.
[32] Preparation and Assay of Phosphorylating Submitochondrial Particles
By C. L. W~mKINS and A. L. LEHNINGER Extraction of rat liver mitochondria with digitonin gives rise to submitochondrial fragments which possess intact respiratory chains and couple the phosphorylation of ADP with the oxidation of v-flhydroxybutyrate. Preparation of Submitochondrial Particles 1 Twice-washed mitochondria prepared by the method described by Hogeboom 2 are suspended evenly in 6.0 ml of chilled 0.05M sucrose for each 100 g. of liver tissue used. After the total volume of this mitochondrial suspension is determined, 0.66 vol. of a chilled 2 ~ solution of recrystallizeds digitonin (Fisher Scientific Co.) in 0.05M sucrose is 1T. M. Devlin and A. L. Lehninger, J. Biol. Chem. 233, 1586 (1958). *G. H. Hogeboom, Vol. I [3]. s Commercial digitonin should be recrystallized from hot ethanol; some batches of commercial preparations contain heavy metal ions.
266
E~ZYMES OF PHOSPHATE METABOLISM
[32]
added dropwise with continuous stirring at 0 ° to yield a final digitonin concentration of 0.8~. The suspension is then maintained at 0 ° for 20 minutes at which time sufficient chilled 0.25 M sucrose is added to reduce the digitonin concentration to 0.25%. The mixture is transferred to chilled tubes and centrifuged at 20,000 X g at 2 ° for 20 minutes to remove unfragmented mitochondria. The supernatant solution is removed so that the upper, loosely packed layer is also decanted; this supernatant is recentrifuged at 80,000 X g for 25 minutes. The supernatant solution is discarded, and the pellet, which contains the active fragments, is suspended in cold distilled water. The pellet obtained from the mitechondria of 50 g. of rat liver is suspended to a final volume of 8 ml. and normally contains 0.8 to 1.2 mg. of nitrogen per milliliter. This suspension may be used directly in the phosphorylation assay system 1,~ or for studies of the ATP-P~ 82 exchange,5 the ATPase activity, e or the ATPADP exchange activity. 7 Oxidation Phosphorylation Assay Method
Principle. In the presence of ADP and inorganic phosphate, the submitochondrial particles will oxidize D-fl-hydroxybutyric acid quantitatively to acetoacetic acid with coupled production of ATP. Although P: 0 ratios of 1.5 to 2.0 are obtained with this system, the addition of a hexokinase-glucose phosphate "trap" has made it possible to obtain routinely P/O ratios above 2.0 (2.0 to 2.5). Acetoacetate is determined by a modification of the sensitive and specific method of Walker 8 which involves coupling with diazotized p-nitroaniline to produce the N,N'di(-p-nitrophenyl-) C-acetylformazan, which shows characteristic absorption at 450 m~. ATP production is estimated by employing P32-1abeled inorganic phosphate in the reaction medium and measuring the appearance of glucose-6-P~2. This is done by separation of the residual PS2-1abeled inorganic phosphate and labeled glucose-6-P by reaction of the inorganic phosphate with ammonium molybdate followed by quantitative extraction of the phosphomolybdate complex with an isobutanol-benzene mixture2 The labeled glucose-6-P is present in the aqueous phase. If the hexokinase-glucose system is not used, labeled ATP will be present in the aqueous phase. C. 5C. C. 7C. 8p. ' S.
Cooper and A. L. Lehninger, J. Biol. Chem. 219, 489 (1956). Cooper and A. L. Lehninger, I. Biol. Chem. 224, 561 (1957). Cooper and A. L. Lehninger, J. Biol. Chem. 224, 547 (1957). L. Wadkins and A. L. Lehninger, J. Biol. Chem. 233, 1589 (1958). G. Walker, Bioehem. J. 58, 699 (1954). O. Nielson and A. L. Lehninger, J. Biol. Chem. 215, 555 (1955).
[32]
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
267
Reagents 0.2 M DL-fl-hydroxybutyrie acid, pH 6.5. 0.1 M histidine buffer, pH 6.5. 0.3M potassium phosphate, pH 6.5. 0.025M adenosine diphosphate, pH 6.5. 0.19 M trichloroacetie acid. Hexokinase (Sigma Type V 500,000 K.M. units/g.), 10.0 mg./ml, in 0.25M glucose (combined reagent). Pyrophosphate-free P3~-labeled orthophosphate, 5,000,000 c.p.m./ml. Digitonin particles, 0.8 to 1.2 mg. of nitrogen per milliliter. Reaction Medium. The reaction medium contained the following components, in the final concentrations given: 0.1 ml. of DL-fl-hydroxybutyric acid, 0.02M; 0.1 ml. of histidine buffer, pH 6.5, 0.01M; 0.1 ml. of K-phosphate, pH 6.5, 0.03M; 0.1 ml. of P3~-orthophosphate, 500,000 c.p.m., containing 0.1 ml. of adenosine diphosphate, 0.0025M; 0.1 ml. of glueose-hexokinase, 0.025 M, yielding 1 mg. of hexokinase per milliliter of reaction medium; water to a final volume of 1.0 ml.; and 0.2 ml. of digitonin particles, 150 to 200 ~g. of nitrogen per milliliter. Procedure. Enzyme is added last to start the reaction. The reaction is usually carried out for 20 minutes at 20 ° to 22 ° with shaking in air and is terminated by addition of 3.5 ml. of 0.19M trichloroacetic acid. After removal of protein by centrifugation, aliquots of the supernatant liquid are used for ATP ~2 and acetoacetate analysis. Acetoacetate Analysis Reagents p-Nitroaniline, 0.05~ in 0.05 N HC1. Sodium nitrite, 0.5% (must be prepared just before use). Acetmte buffer, 1.0M, pH 5.7. Hydrochloric acid, 5.0N. Sodium acetate, 0.2M. Diazo reagent. This reagent is prepared just before use by adding 15 ml. of sodium nitrite to 100 ml. of p-nitroaniline and then cooling to 2 °. Then 35 ml. of 0.2 M sodium acetate is added. Preparation o] Samples. Deproteinized reaction system (1.5 ml.) containing 0.15M trichloroaeetie acid is mixed with 1.5 ml. of 1.0M acetate buffer. The pH of the system at this point must be 4.9 to 5.0. Six milliliters of the freshly prepared diazo reagent is added, and the mixture is kept at 22 ° for 40 minutes followed by the addition of 2.0 ml.
268
ENZYMES OF PHOSPHATE METABOLISM
[32]
of 5 N HCl. The system is extracted with 8.0 ml. of ethyl acetate. Three milliliters of ethyl acetate phase is placed in a l-era, cuvette, and the extinction is determined at 450 m~ in a Beckman DU spectrophotometer. An optical density of 1.0 corresponds to 840 millimicromoles of acetoacetate in the original enzymatic assay system. It is unnecessary to use acetoacetate, an unstable compound, as working standard, since the extinction coefficient of the product of the reaction with diazotized P-nitroaniline has been found to be constant under the conditions used here.8
Analysis ]or Phosphate Uptake~ Reagents Isobutanol-benzene reagent. This is prepared by shaking equal volumes of water-saturated isobutanol and water-saturated benzene and allowing the mixture to stand until the excess water separates. Ammonium molybdate-H2SO4 reagent, 5 ~ ammonium molybdate in 4N sulfuric acid. Acetone.
Procedure. One milliliter of acetone and a 0.5-ml. sample of deproteinized reaction medium are mixed in a 40-ml. glass-stoppered shaking tube and allowed to stand for 10 minutes. Then 2.0 ml. of water and 7.0 ml. of isobutanol-benzene reagent are added, and the mixture is shaken vigorously for 30 seconds. Next 1.5 ml. of acid-molybdate reagent is added and mixed by gentle swirling. This mixture is allowed to stand for 5 minutes. The mixture is then shaken vigorously for 30 seconds and centrifuged at 1500 r.p.m, for 3 to 4 minutes. The bottom (aqueous) phase is removed and filtered through Whatman No. 50 paper into a second shaking tube containing 4.0 ml. of isobutanol-benzene reagent. This mixture is shaken vigorously and centrifuged at 1500 r.p.m, for 4 minutes. The aqueous phase is withdrawn and filtered through paper as before. The radioactivity of this sample is now determined by counting wet 1.0 ml. with constant geometry. The net formation of ATP during the reaction is calculated from the radioactivity, A, of the extracted sample (counts per minute), the specific radioactivity, B, of the inorganic phosphate of the original reaction system (counts per minute per micromole), and the dilution introduced during the extraction procedure: A 1.0 + 3.5 q- Aureoles ATP = -~ X 4 X 0.5
[32]
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
The P/O ratio can then be expressed
269
as
-{-A~moles A T P P/O -- ~_ ~moles acetoacetate Properties of the Phosphorylation System Specificity.n-fl-Hydroxybutyric acid, succinic acid, and ascorbate plus external cytochrome c are the only substrates whose oxidation is coupled to the synthesis of A T P in these preparations, but the efficiency with succinate is low (P/0 ----0.2). A D P is the only nucleotide which will serve as a phosphate acceptor. Stability. When aged at 30 °, approximately 50~ of the oxidationcoupled phosphorylation of ADP is lost in 60 minutes. At 2 °, 50% loss occurs in 24 hours. Freezing and thawing completely destroy enzymatic activity. Addition of bovine serum albumin to the test medium (2 mg./ ml.) will restore some of the activity of aged preparations. Activators and Inhibitors. The addition of metal ions is not required for optimal P / 0 ratios, although the submitochondrial particles do contain bound Mg +÷. Bovine serum albumin (2 mg./ml.) usually improves the P/O ratio and will prevent uncoupling by long-chain fatty acids. 2,4-Dinitrophenol, gramicidin, Dicumarol, arsenate, and azide uncouple at the same concentrations found to be effective with intact mitochondria, but thyroxine and its analogs and Ca ++ do not act as uncoupling agents. Optimal P/O ratios are obtained at pH 6.5.
"Partial Reactions" of Oxidative Phosphorylation The submitochondrial particles catalyze two exchange reactions which are considered to represent intermediate steps in the coupling process. The ATP-P~ 3s exchange is thought to be a manifestation of those steps involved in the uptake of inorganic phosphate with the ultimate synthesis of ATP, 5 and the ATP-ADP (C 14) exchange reaction is considered to represent the final transphosphorylation reaction whereby ADP reacts with a phosphorylated intermediate to form ATP. 7
Assay Method for the A T P - P ~ Exchange Reaction 5
Principle. The assay of this reaction involves incubation of ps2_ labeled inorganic orthophosphate, ATP, and the submitochondrial particles and, after termination of the reaction with trichloroacetic acid, estimation of the pa2 content of the A T P of the reaction medium. This is accomplished by isobutanol-benzene extraction of the phosphomolybdate complex which leaves the labeled A T P in the aqueous phase. Determination of the radioactivity of the aqueous phase makes it possible to calculate the extent of incorporation of inorganic phosphate into ATP.
270
ENZYMES OF PHOSPHATE METABOLISM
[32]
Procedure. Optimal conditions for assaying the ATP-P~ "~2 exchange reaction consist of a reaction system containing 6 mieromoles of ATP, 10 micromoles of sodium or potassium phosphate containing approximately 600,000 c.p.m, of p~.~2, 10 micromoles of histidine or imidazole buffer, pH 6.5, and water to a volume of 0.9 ml. The reaction is started by addition of 0.1 ml. of enzyme suspension. The system is incubated at 25 ° for 20 minutes and stopped by addition of 1.0 ml. of 13% trichloroacetie acid. The protein is removed by centrifugation, and 0.5 ml. of supernatant liquid is used for analysis of P32-1abeled ATP by the method described in the section on oxidative phosphorylation. The extent of incorporation of p32 into ATP is calculated from the determination of the radioactivity, A, of the extracted sample (counts per minute), the specific activity, B, of the inorganic phosphate of the original reaction system (counts per minute per micromole), and correction for the dilution imposed during the extraction procedure. A
-AP~ 32 = ~ × 4
X
1.0 + 1.0 = umoles pa2 exchanged 0.5
Properties of the ATP-P¢ 32Exchange Reaction
Rate o] Reaction. Under the respective optimal conditions, equivalent amounts of the enzyme catalyze the ATP-P~ ~ exchange reaction at a rate approximately 5% that of respiration-linked phosphorylation. However, the rate of the ATP-P~ ~2 exchange reaction is markedly dependent on the A T P / A D P ratio; as this ratio becomes smaller, the exchange rate approaches zero. The extent to which the ATP-P~ 32 exchange occurs during respiration-linked phosphorylation has been found to be negligible. In the exchange reaction the incorporation of P32-1abeled inorganic phosphate into ATP occurs almost linearly. When higher concentrations of enzyme are employed, approach to isotopic equilibrium can be observed. An accurate estimation of the initial reaction rate can be made when the conditions described above are employed. Stability. The stability of the ATP-P~ 32 exchange system is quite similar to that of respiration-coupled phosphorylation. Approximately 50% loss of activity occurs in 24 hours at 2 ° . Freezing and thawing completely inactivate. Inhibitors and Activators. The exchange activity is completely inhibited by 2,4-dinitrophenol, Dicumarol, gramicidin, arsenate, and azide at concentrations that also completely uncouple oxidative phosphorylation. The addition of metal ions is not required. Serum albumin (2 mg./ ml.) will enhance the rate of exchange and will also prevent inhibition by long-chain fatty acids.
[32]
PHOSPHORYI~TING SUBM1TOCHONDRIAL PARTICLES
271
Assay Method for the ATP-ADP Exchange Reaction 7
Principle. This reaction may be assayed by incubation of either P ~2labeled ADP 5 or C14-1abeled ADP, 1° unlabeled ATP, and the digitonin enzyme. After the reaction is stopped, the nucleotides are separated by paper chromatography and their specific activities determined after elution from the paper. Occurrence of the exchange is indicated by the amount of isotope found in the ATP. Procedure. The A T P - A D P exchange reaction is carried out in a reaction system containing 3.0 micromoles of ATP, 3.0 micromoles of ADP containing 30,000 to 35,000 c.p.m, of C14-ADP, 5.0 micromoles of histidine or imidazole buffer, pH 6.5, in 0.45-ml. volume. The enzyme suspension (0.05 ml. ; 0.8 to 1.2 mg. of nitrogen per milliliter) is added to start the reaction which is allowed to proceed for 10 minutes at 25 °. The reaction is stopped by addition of 0.05 ml. of 65% trichloroacetic acid, and the protein is removed bY centrifugation. Fifty microliters of the supernatant solution is placed on a sheet of acid-washed Whatman No. 1 paper ( 1 8 X 4 7 cm.), on a line approximately 8 cm. from the top. Four such spots can be placed on one sheet. The sheets are placed in jars for descending chromatography. The chromatogram is developed by a reagent consisting of 66 ml. of isobutyric acid, 1 ml. of concentrated ammonium hydroxide, and 33 ml. of water. Twenty hours is usually sufficient to produce quantitative separation of the ATP and ADP during which time the solvent front runs off the bottom of the paper. The nucleotides are located by scanning the dried sheets with an ultraviolet Mineralight. 11 In a typical chromatographic separation of a mixture containing ATP, ADP, and AMP, the leading edges of the spots corresponding to these nucleotides were l0 cm., 16 cm., and 24 cm., respectively, from the origin. The areas containing the nucleotides are marked, cut out, and sectioned in 1-cm. squares. The squares of paper are immersed in 3.0 ml. of water for 15 minutes with occasional swirling of the container. One milliliter is placed on a planchef, evaporated to dryness, and counted by means of a gas-flow windowle.~s counter. The extent to which the exchange has occurred can be calculated by the relationship:
C.p.m.(ATP)
C.p.m.cADe)/micromoles ADP
X 33 ---- Micromoles ATP exchanged
The dilution factor for this operation is 33 [3 X (0.50-[-0.05)/0.05]. '°Adenosine diphosphate-8-C 14, Schwartz Laboratories, Inc., Mount Vernon, New York. ~IMineralight Model SL 2537, Ultra-Violet Products, Inc., San Gabriel, California.
272
ENZYMES OF PHOSPHATE METABOLISM
[33]
Properties of the A T P - A D P Exchange Reaction
Specificity. The exchange reaction occurs only with ATP and ADP. Stability. The exchange activity is quite stable; 50% loss occurs in 1 week when an aqueous suspension of the submitochondrial particles is stored at 2 ° . Inhibitors. The A T P - A D P exchange reaction is inhibited 50% by 5 X 10-SM phenylmercuric acetate and by 1 X 10-'M p-chloromercuribenzoic acid. This inhibition is reversed by addition of cysteine or reduced glutathione. The exchange activity of fresh submitochondrial particles is inhibited by low concentrations of D N P and Dicumarol; these agents do not inhibit with aged enzyme preparations. Other Reactions The digitonin particles also catalyze DNP-stimulated ATPase activity 6 and the exchange Of 0 TM between H20 of the medium and inorganic phosphate and the terminal phosphate of ATP.
[33] Preparation and Assay of Phosphorylating Submitochondrial Particles: Sonicated Mitochondria 1
By W. WAYNE KIELLEY
Assay Method Principle. Although standard manometric and colorimetric techniques can be used for measuring oxygen and phosphate uptake with particles obtained by sonic disintegration of mitochondria, ~ it has been found convenient and somewhat more precise to utilize the oxygen electrode for measuring oxygen uptake with this material. The Clark oxygen electrode 3 as used here measures the decrease in dissolved oxygen. As described, this involves amounts of the order of 0.2 to 0.3 ~atom of oxygen. Therefore, an equally sensitive method for phosphate uptake, the incorporation of p82 into organic phosphate, is required. Reagents 0.2 M PO,, pH 7.0. 0.1 M MgC12. t The methods described here are taken from W. W. Kielley and J. R. Bronk, J. Biol. Chem. 230, 521 (1958). ' W. C. McMurray, G. F. Maley, and H. A. Lardy, J. Biol. Chem. 230, 219 (1958). s L. C. Clark, Jr., R. Wolf, G. Granger, and Z. Taylor, J. Appl. Physiol. 6, 189 (1953).
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
265
centrifugation, the enzyme is recovered without loss in the supernatant fluid, whereas 70% of the protein is discarded in the precipitate. Properties Enzyme fractions stored for weeks at --13 ° , often with repeated thawing and refreezing, show no signs of deterioration. Fractions 2 and 3, retested after 6 to 8 weeks, show no detectable loss in activity. The optimum pH of the reaction, determined in Tris buffers in the presence of a severalfold excess of acetokinase, is found to be near 7.2. The rates at pH 6.2, 6.7, 7.5, and 8.2 have been found to be 45, 75, 83, and 73%, respectively, of the rate at pH 7.2. At pH 7.2 the rate in phosphate buffer is about 25~6 slower than in Tris buffer. Ammonium sulfate stimulates the reaction rate of enzyme fraction 6; the rate in the presence of 0.04 M ammonium sulfate appears maximal and almost three times the rate in its absence. When the enzyme solubilized by ammonium sulfate was studied, the addition of ammonium sulfate did not stimulate the rate, although the concentration of the salt was only 0.002 M. Fluoride (5 X 10-* M) completely inhibits the enzyme; 2,4-dinitrophenol (3 X 10-4 M) does not inhibit. Magnesium is essential to the reaction.
[32] Preparation and Assay of Phosphorylating Submitochondrial Particles
By C. L. W~mKINS and A. L. LEHNINGER Extraction of rat liver mitochondria with digitonin gives rise to submitochondrial fragments which possess intact respiratory chains and couple the phosphorylation of ADP with the oxidation of v-flhydroxybutyrate. Preparation of Submitochondrial Particles 1 Twice-washed mitochondria prepared by the method described by Hogeboom 2 are suspended evenly in 6.0 ml of chilled 0.05M sucrose for each 100 g. of liver tissue used. After the total volume of this mitochondrial suspension is determined, 0.66 vol. of a chilled 2 ~ solution of recrystallizeds digitonin (Fisher Scientific Co.) in 0.05M sucrose is 1T. M. Devlin and A. L. Lehninger, J. Biol. Chem. 233, 1586 (1958). *G. H. Hogeboom, Vol. I [3]. s Commercial digitonin should be recrystallized from hot ethanol; some batches of commercial preparations contain heavy metal ions.
266
E~ZYMES OF PHOSPHATE METABOLISM
[32]
added dropwise with continuous stirring at 0 ° to yield a final digitonin concentration of 0.8~. The suspension is then maintained at 0 ° for 20 minutes at which time sufficient chilled 0.25 M sucrose is added to reduce the digitonin concentration to 0.25%. The mixture is transferred to chilled tubes and centrifuged at 20,000 X g at 2 ° for 20 minutes to remove unfragmented mitochondria. The supernatant solution is removed so that the upper, loosely packed layer is also decanted; this supernatant is recentrifuged at 80,000 X g for 25 minutes. The supernatant solution is discarded, and the pellet, which contains the active fragments, is suspended in cold distilled water. The pellet obtained from the mitechondria of 50 g. of rat liver is suspended to a final volume of 8 ml. and normally contains 0.8 to 1.2 mg. of nitrogen per milliliter. This suspension may be used directly in the phosphorylation assay system 1,~ or for studies of the ATP-P~ 82 exchange,5 the ATPase activity, e or the ATPADP exchange activity. 7 Oxidation Phosphorylation Assay Method
Principle. In the presence of ADP and inorganic phosphate, the submitochondrial particles will oxidize D-fl-hydroxybutyric acid quantitatively to acetoacetic acid with coupled production of ATP. Although P: 0 ratios of 1.5 to 2.0 are obtained with this system, the addition of a hexokinase-glucose phosphate "trap" has made it possible to obtain routinely P/O ratios above 2.0 (2.0 to 2.5). Acetoacetate is determined by a modification of the sensitive and specific method of Walker 8 which involves coupling with diazotized p-nitroaniline to produce the N,N'di(-p-nitrophenyl-) C-acetylformazan, which shows characteristic absorption at 450 m~. ATP production is estimated by employing P32-1abeled inorganic phosphate in the reaction medium and measuring the appearance of glucose-6-P~2. This is done by separation of the residual PS2-1abeled inorganic phosphate and labeled glucose-6-P by reaction of the inorganic phosphate with ammonium molybdate followed by quantitative extraction of the phosphomolybdate complex with an isobutanol-benzene mixture2 The labeled glucose-6-P is present in the aqueous phase. If the hexokinase-glucose system is not used, labeled ATP will be present in the aqueous phase. C. 5C. C. 7C. 8p. ' S.
Cooper and A. L. Lehninger, J. Biol. Chem. 219, 489 (1956). Cooper and A. L. Lehninger, I. Biol. Chem. 224, 561 (1957). Cooper and A. L. Lehninger, J. Biol. Chem. 224, 547 (1957). L. Wadkins and A. L. Lehninger, J. Biol. Chem. 233, 1589 (1958). G. Walker, Bioehem. J. 58, 699 (1954). O. Nielson and A. L. Lehninger, J. Biol. Chem. 215, 555 (1955).
[32]
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
267
Reagents 0.2 M DL-fl-hydroxybutyrie acid, pH 6.5. 0.1 M histidine buffer, pH 6.5. 0.3M potassium phosphate, pH 6.5. 0.025M adenosine diphosphate, pH 6.5. 0.19 M trichloroacetie acid. Hexokinase (Sigma Type V 500,000 K.M. units/g.), 10.0 mg./ml, in 0.25M glucose (combined reagent). Pyrophosphate-free P3~-labeled orthophosphate, 5,000,000 c.p.m./ml. Digitonin particles, 0.8 to 1.2 mg. of nitrogen per milliliter. Reaction Medium. The reaction medium contained the following components, in the final concentrations given: 0.1 ml. of DL-fl-hydroxybutyric acid, 0.02M; 0.1 ml. of histidine buffer, pH 6.5, 0.01M; 0.1 ml. of K-phosphate, pH 6.5, 0.03M; 0.1 ml. of P3~-orthophosphate, 500,000 c.p.m., containing 0.1 ml. of adenosine diphosphate, 0.0025M; 0.1 ml. of glueose-hexokinase, 0.025 M, yielding 1 mg. of hexokinase per milliliter of reaction medium; water to a final volume of 1.0 ml.; and 0.2 ml. of digitonin particles, 150 to 200 ~g. of nitrogen per milliliter. Procedure. Enzyme is added last to start the reaction. The reaction is usually carried out for 20 minutes at 20 ° to 22 ° with shaking in air and is terminated by addition of 3.5 ml. of 0.19M trichloroacetic acid. After removal of protein by centrifugation, aliquots of the supernatant liquid are used for ATP ~2 and acetoacetate analysis. Acetoacetate Analysis Reagents p-Nitroaniline, 0.05~ in 0.05 N HC1. Sodium nitrite, 0.5% (must be prepared just before use). Acetmte buffer, 1.0M, pH 5.7. Hydrochloric acid, 5.0N. Sodium acetate, 0.2M. Diazo reagent. This reagent is prepared just before use by adding 15 ml. of sodium nitrite to 100 ml. of p-nitroaniline and then cooling to 2 °. Then 35 ml. of 0.2 M sodium acetate is added. Preparation o] Samples. Deproteinized reaction system (1.5 ml.) containing 0.15M trichloroaeetie acid is mixed with 1.5 ml. of 1.0M acetate buffer. The pH of the system at this point must be 4.9 to 5.0. Six milliliters of the freshly prepared diazo reagent is added, and the mixture is kept at 22 ° for 40 minutes followed by the addition of 2.0 ml.
268
ENZYMES OF PHOSPHATE METABOLISM
[32]
of 5 N HCl. The system is extracted with 8.0 ml. of ethyl acetate. Three milliliters of ethyl acetate phase is placed in a l-era, cuvette, and the extinction is determined at 450 m~ in a Beckman DU spectrophotometer. An optical density of 1.0 corresponds to 840 millimicromoles of acetoacetate in the original enzymatic assay system. It is unnecessary to use acetoacetate, an unstable compound, as working standard, since the extinction coefficient of the product of the reaction with diazotized P-nitroaniline has been found to be constant under the conditions used here.8
Analysis ]or Phosphate Uptake~ Reagents Isobutanol-benzene reagent. This is prepared by shaking equal volumes of water-saturated isobutanol and water-saturated benzene and allowing the mixture to stand until the excess water separates. Ammonium molybdate-H2SO4 reagent, 5 ~ ammonium molybdate in 4N sulfuric acid. Acetone.
Procedure. One milliliter of acetone and a 0.5-ml. sample of deproteinized reaction medium are mixed in a 40-ml. glass-stoppered shaking tube and allowed to stand for 10 minutes. Then 2.0 ml. of water and 7.0 ml. of isobutanol-benzene reagent are added, and the mixture is shaken vigorously for 30 seconds. Next 1.5 ml. of acid-molybdate reagent is added and mixed by gentle swirling. This mixture is allowed to stand for 5 minutes. The mixture is then shaken vigorously for 30 seconds and centrifuged at 1500 r.p.m, for 3 to 4 minutes. The bottom (aqueous) phase is removed and filtered through Whatman No. 50 paper into a second shaking tube containing 4.0 ml. of isobutanol-benzene reagent. This mixture is shaken vigorously and centrifuged at 1500 r.p.m, for 4 minutes. The aqueous phase is withdrawn and filtered through paper as before. The radioactivity of this sample is now determined by counting wet 1.0 ml. with constant geometry. The net formation of ATP during the reaction is calculated from the radioactivity, A, of the extracted sample (counts per minute), the specific radioactivity, B, of the inorganic phosphate of the original reaction system (counts per minute per micromole), and the dilution introduced during the extraction procedure: A 1.0 + 3.5 q- Aureoles ATP = -~ X 4 X 0.5
[32]
PHOSPHORYLATING SUBMITOCHONDRIAL PARTICLES
The P/O ratio can then be expressed
269
as
-{-A~moles A T P P/O -- ~_ ~moles acetoacetate Properties of the Phosphorylation System Specificity.n-fl-Hydroxybutyric acid, succinic acid, and ascorbate plus external cytochrome c are the only substrates whose oxidation is coupled to the synthesis of A T P in these preparations, but the efficiency with succinate is low (P/0 ----0.2). A D P is the only nucleotide which will serve as a phosphate acceptor. Stability. When aged at 30 °, approximately 50~ of the oxidationcoupled phosphorylation of ADP is lost in 60 minutes. At 2 °, 50% loss occurs in 24 hours. Freezing and thawing completely destroy enzymatic activity. Addition of bovine serum albumin to the test medium (2 mg./ ml.) will restore some of the activity of aged preparations. Activators and Inhibitors. The addition of metal ions is not required for optimal P / 0 ratios, although the submitochondrial particles do contain bound Mg +÷. Bovine serum albumin (2 mg./ml.) usually improves the P/O ratio and will prevent uncoupling by long-chain fatty acids. 2,4-Dinitrophenol, gramicidin, Dicumarol, arsenate, and azide uncouple at the same concentrations found to be effective with intact mitochondria, but thyroxine and its analogs and Ca ++ do not act as uncoupling agents. Optimal P/O ratios are obtained at pH 6.5.
"Partial Reactions" of Oxidative Phosphorylation The submitochondrial particles catalyze two exchange reactions which are considered to represent intermediate steps in the coupling process. The ATP-P~ 3s exchange is thought to be a manifestation of those steps involved in the uptake of inorganic phosphate with the ultimate synthesis of ATP, 5 and the ATP-ADP (C 14) exchange reaction is considered to represent the final transphosphorylation reaction whereby ADP reacts with a phosphorylated intermediate to form ATP. 7
Assay Method for the A T P - P ~ Exchange Reaction 5
Principle. The assay of this reaction involves incubation of ps2_ labeled inorganic orthophosphate, ATP, and the submitochondrial particles and, after termination of the reaction with trichloroacetic acid, estimation of the pa2 content of the A T P of the reaction medium. This is accomplished by isobutanol-benzene extraction of the phosphomolybdate complex which leaves the labeled A T P in the aqueous phase. Determination of the radioactivity of the aqueous phase makes it possible to calculate the extent of incorporation of inorganic phosphate into ATP.
270
ENZYMES OF PHOSPHATE METABOLISM
[32]
Procedure. Optimal conditions for assaying the ATP-P~ "~2 exchange reaction consist of a reaction system containing 6 mieromoles of ATP, 10 micromoles of sodium or potassium phosphate containing approximately 600,000 c.p.m, of p~.~2, 10 micromoles of histidine or imidazole buffer, pH 6.5, and water to a volume of 0.9 ml. The reaction is started by addition of 0.1 ml. of enzyme suspension. The system is incubated at 25 ° for 20 minutes and stopped by addition of 1.0 ml. of 13% trichloroacetie acid. The protein is removed by centrifugation, and 0.5 ml. of supernatant liquid is used for analysis of P32-1abeled ATP by the method described in the section on oxidative phosphorylation. The extent of incorporation of p32 into ATP is calculated from the determination of the radioactivity, A, of the extracted sample (counts per minute), the specific activity, B, of the inorganic phosphate of the original reaction system (counts per minute per micromole), and correction for the dilution imposed during the extraction procedure. A
-AP~ 32 = ~ × 4
X
1.0 + 1.0 = umoles pa2 exchanged 0.5
Properties of the ATP-P¢ 32Exchange Reaction
Rate o] Reaction. Under the respective optimal conditions, equivalent amounts of the enzyme catalyze the ATP-P~ ~ exchange reaction at a rate approximately 5% that of respiration-linked phosphorylation. However, the rate of the ATP-P~ ~2 exchange reaction is markedly dependent on the A T P / A D P ratio; as this ratio becomes smaller, the exchange rate approaches zero. The extent to which the ATP-P~ 32 exchange occurs during respiration-linked phosphorylation has been found to be negligible. In the exchange reaction the incorporation of P32-1abeled inorganic phosphate into ATP occurs almost linearly. When higher concentrations of enzyme are employed, approach to isotopic equilibrium can be observed. An accurate estimation of the initial reaction rate can be made when the conditions described above are employed. Stability. The stability of the ATP-P~ 32 exchange system is quite similar to that of respiration-coupled phosphorylation. Approximately 50% loss of activity occurs in 24 hours at 2 ° . Freezing and thawing completely inactivate. Inhibitors and Activators. The exchange activity is completely inhibited by 2,4-dinitrophenol, Dicumarol, gramicidin, arsenate, and azide at concentrations that also completely uncouple oxidative phosphorylation. The addition of metal ions is not required. Serum albumin (2 mg./ ml.) will enhance the rate of exchange and will also prevent inhibition by long-chain fatty acids.
[32]
PHOSPHORYI~TING SUBM1TOCHONDRIAL PARTICLES
271
Assay Method for the ATP-ADP Exchange Reaction 7
Principle. This reaction may be assayed by incubation of either P ~2labeled ADP 5 or C14-1abeled ADP, 1° unlabeled ATP, and the digitonin enzyme. After the reaction is stopped, the nucleotides are separated by paper chromatography and their specific activities determined after elution from the paper. Occurrence of the exchange is indicated by the amount of isotope found in the ATP. Procedure. The A T P - A D P exchange reaction is carried out in a reaction system containing 3.0 micromoles of ATP, 3.0 micromoles of ADP containing 30,000 to 35,000 c.p.m, of C14-ADP, 5.0 micromoles of histidine or imidazole buffer, pH 6.5, in 0.45-ml. volume. The enzyme suspension (0.05 ml. ; 0.8 to 1.2 mg. of nitrogen per milliliter) is added to start the reaction which is allowed to proceed for 10 minutes at 25 °. The reaction is stopped by addition of 0.05 ml. of 65% trichloroacetic acid, and the protein is removed bY centrifugation. Fifty microliters of the supernatant solution is placed on a sheet of acid-washed Whatman No. 1 paper ( 1 8 X 4 7 cm.), on a line approximately 8 cm. from the top. Four such spots can be placed on one sheet. The sheets are placed in jars for descending chromatography. The chromatogram is developed by a reagent consisting of 66 ml. of isobutyric acid, 1 ml. of concentrated ammonium hydroxide, and 33 ml. of water. Twenty hours is usually sufficient to produce quantitative separation of the ATP and ADP during which time the solvent front runs off the bottom of the paper. The nucleotides are located by scanning the dried sheets with an ultraviolet Mineralight. 11 In a typical chromatographic separation of a mixture containing ATP, ADP, and AMP, the leading edges of the spots corresponding to these nucleotides were l0 cm., 16 cm., and 24 cm., respectively, from the origin. The areas containing the nucleotides are marked, cut out, and sectioned in 1-cm. squares. The squares of paper are immersed in 3.0 ml. of water for 15 minutes with occasional swirling of the container. One milliliter is placed on a planchef, evaporated to dryness, and counted by means of a gas-flow windowle.~s counter. The extent to which the exchange has occurred can be calculated by the relationship:
C.p.m.(ATP)
C.p.m.cADe)/micromoles ADP
X 33 ---- Micromoles ATP exchanged
The dilution factor for this operation is 33 [3 X (0.50-[-0.05)/0.05]. '°Adenosine diphosphate-8-C 14, Schwartz Laboratories, Inc., Mount Vernon, New York. ~IMineralight Model SL 2537, Ultra-Violet Products, Inc., San Gabriel, California.
272
ENZYMES OF PHOSPHATE METABOLISM
[33]
Properties of the A T P - A D P Exchange Reaction
Specificity. The exchange reaction occurs only with ATP and ADP. Stability. The exchange activity is quite stable; 50% loss occurs in 1 week when an aqueous suspension of the submitochondrial particles is stored at 2 ° . Inhibitors. The A T P - A D P exchange reaction is inhibited 50% by 5 X 10-SM phenylmercuric acetate and by 1 X 10-'M p-chloromercuribenzoic acid. This inhibition is reversed by addition of cysteine or reduced glutathione. The exchange activity of fresh submitochondrial particles is inhibited by low concentrations of D N P and Dicumarol; these agents do not inhibit with aged enzyme preparations. Other Reactions The digitonin particles also catalyze DNP-stimulated ATPase activity 6 and the exchange Of 0 TM between H20 of the medium and inorganic phosphate and the terminal phosphate of ATP.
[33] Preparation and Assay of Phosphorylating Submitochondrial Particles: Sonicated Mitochondria 1
By W. WAYNE KIELLEY
Assay Method Principle. Although standard manometric and colorimetric techniques can be used for measuring oxygen and phosphate uptake with particles obtained by sonic disintegration of mitochondria, ~ it has been found convenient and somewhat more precise to utilize the oxygen electrode for measuring oxygen uptake with this material. The Clark oxygen electrode 3 as used here measures the decrease in dissolved oxygen. As described, this involves amounts of the order of 0.2 to 0.3 ~atom of oxygen. Therefore, an equally sensitive method for phosphate uptake, the incorporation of p82 into organic phosphate, is required. Reagents 0.2 M PO,, pH 7.0. 0.1 M MgC12. t The methods described here are taken from W. W. Kielley and J. R. Bronk, J. Biol. Chem. 230, 521 (1958). ' W. C. McMurray, G. F. Maley, and H. A. Lardy, J. Biol. Chem. 230, 219 (1958). s L. C. Clark, Jr., R. Wolf, G. Granger, and Z. Taylor, J. Appl. Physiol. 6, 189 (1953).