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A single-extract method for the determination of oxidized and reduced diphosphopyridine nucleotide in rat liver
308
LETTERS
TO
THE
EDITORS
basis of ultraviolet spectrophotometry, 1.4 mg. was isolated, which represent 0.5oJc of the administered cortisol. By a similar procedure, late human pregnancy urine (a a-day urine collection) was extracted and the extract chromatographed on paper. The most polar reducing zone was acetylated and rechromatographed on paper. Several cr,fl-unsaturated ketonic steroids were observed under ultraviolet light. The major reducing zone was eluted. On the basis of ultraviolet spectrophotometry, 300 pg. of material was present. The material could not be crystallized, but the infrared analysis and the absorption spectrum in sulfuric acid indicated that 6p-hydroxycortisol was present. This is the first instance of in. &JO 6&hydroxylation in the human. Further studies on the excretion of G&hydroxycortisol in various pathological and stress conditions are being done to establish the biological significance of the 6&hydroxylation in vivo. REFERENCES 1. BURSTEIN,
S., DORFMAN,
R. I., AND NADEL,
E. M., Federation
Proc. 13, 188
(1954). 2. LIEBERMAN,
S., FUKUSHIMA,
D. K., AND DOBRINER,
K., J. BioZ. Chem. 182,299
(1950). 3. BURSTEIN,
S., AND DORFMAN,
R. I., J. Biol. Chem. !XMf, 607 (1954).
Worcester Foundation for Experimental Biology, Shrewsbuy, Massachusetts National Cancer Institute, National Institutes of Health, U. S. Department of Health, Education and Welfare, Bethesda, Maryland Received September dS,196+$ A Single-Extract
SHLOMO BURSTEIN RALPH I. DORFMAN ELI M. NADEL
Method for the Determination of Oxidized Diphosphopyridine Nucleotide in Rat Liver’
and Reduced
The three methods used most in the past for the determination of DPNz and DPNH in tissues are cumbersome and dependent upon the simultaneous standardization and assay of purity of a DPN preparation (l-4). With these three techniques, values of total rat liver DPN ranging from 500 to 1400 pg./g. fresh weight have been reported (2-6). It is possible that the higher figures are a reflection of the use of DPN standards the purities of which were lower than supposed.3 Since highly purified DPN preparations with known molar extinction co1 This investigation was supported in part by a grant from the U. S. Atomic Energy Commission (Contract No. AT(30-l)-1069) and a grant (A-530) from the National Institutes of Health, U. S. Public Health Service. 2 The following abbreviations are employed: DPN, diphosphopyridine nucleotide; DPNH, dihydrodiphosphopyridine nucleotide; ADH, alcohol dehydrogenase. * Dr. F. Schlenk, personal communication.
LETTERS
TO
THE
EDITORS
309
efficients (7) have been available for some time and a simple, one-enzyme method for the determination of both oxidized and reduced DPN has been described (8), it was decided to apply these findings to reinvestigate the levels of DPN and ‘DPNH in rat liver. Two recently published procedures for the analysis of tissue DPN and DPNH also take advantage of these findings. Thus, Strength et al. (9) reported a method for the determination of rat liver DPN based on extraction with trichloroacetic acid and assay with either lactic dehydrogenase or ADH. Holzer et. al. (10) analyzed rat liver and yeast for their content of DPN and DPNH by extraction with acid and alkali, respectively, and assay with ADH. In the present communication, conditions are described for the simultaneous extraction of both DPN forms from one liver sample. Levels of total liver DPN obtained by this technique average 863 pg./g. fresh weight, a value in good agreement with the data of Jandorf (4) and higher than many figures quoted in the literature (2-6, 10-12). Into each of two Potter-Elvehjem homogenizers are placed 2 ml. of 0.1 M KHzPOl , 2 ml. of a solution containing 0.5% NaHCOr0.5% NazCOa , and 3 ml. of glass-distilled water. The homogenizers are placed in a boiling water bath at 2-min. intervals. Two minutes after inserting the first homogenizer into the water bath, a rat is decapitated and the liver removed immediately and blotted free of blood. A weighed portion of 400-500 mg. of liver (kept at room temperature) is added to the first homogenizer 34 min. after it has been in the bath. After a half minute, the homogenizer is removed, and the liver is homogenized for 30 sec. The homogenizer is then replaced in the water bath for an additional half minute and finally plunged into a container of ice. Another portion of liver is now weighed and added to the second homogenizer 3 min. after it has been in the bath, and the same process is repeated. Both chilled homogenates are diluted quantitatively to 15 ml. with glass-distilled water and centrifuged at 34,850 X 9 for 13 min. in a Spinco ultracentrifuge (model L, rotor No. 30). The yellowish, almost clear supernatants are removed and added to previously chilled test tubes, and the latter are kept cold until used. DPN and DPNH determinations are performed according to Racker (8). One and a half milliliters of extract in a total volume of 3 ml. was found to give good readings with the Beckman DU spectrophotometer at 340 rnp. ADH was obtained from Worthington Biochemical Co. as a suspension in ammonium sulfate; 0.03 ml. of a 1:50 dilution of the enzyme was sufficient to drive both reactions to completion within several minutes. DPN and DPNH were obtained from Sigma. DPNH samples were also prepared chemically (13) and enzymatically with ADH and used without further purification. For a typical DPN determination, the following components are added to the cuvette in order: 1.5 ml. liver extract, 0.3 ml. of 0.1 M pyrophosphate buffer, pH 9.0, 0.1 ml. of 95% ethanol, and water to make a volume of 2.97 ml. After mixing, duplicate determinations of the optical density are made. Following the addition of 0.03 ml. of ADH and careful mixing, the density is recorded at l-mm. intervals until at least two or three unchanged readings are obtained, at which point the reaction is considered complete. For the determination of DPNH, a similar procedure is followed except that 0.3 ml. of 0.1 M KzHPO,-KHzPOI buffer, pH 7.1, is substituted for the pyrophosphate, and 0.1 ml. of 10% redistilled acetaldehyde for the ethanol. The absorption coefficient of 6.22 X lo6 sq. cm./ mole, derived by Horecker and Kornberg (7) for the 340-m& band of DPNH, is used to calculate
310
LETTERS
TO
THE
EDITORS
TABLE Concentration
of Oxidized
and Reduced
I Diphosphopyridine
Nueleotide
in
DPN /DPNH
<2.5
8
4-10
8
51gb (488-552) 415 (30849)
345 (321-418) 338 (308-411)
(79212) 753 (635-867)
(l.lZ2.74) 1.25 (0.94-1.76)
a Wistar males, 110-159 g. b The values represent the average and range. the results. The final reading minus the initial reading, in the case of DPN, and the initial reading minus the final reading, in the case of DPNH, are used to calculate the levels of the two forms of the coenzyme present in the extract. Table I lists liver DPN, DPNH, and DPN/DPNH values of normal male Wistar rats (110-150 g.). It can be seen that the average total figure of 863 ag./g. fresh weight obtained for liver samples placed into the hot medium 2.5 min. or less after decapitation was consistently higher than the mean of 751 pg./g. found for samples taken 4-10 min. after decapitation. This observation corroborates Jandorf’s (4) earlier data. Table I shows that the decline in total coenzyme concentration is due to a decrease in oxidized component. No change occurs in the DPNH concentration during the lo-min. period following decapitation. Accordingly, the DPN/DPNH ratios are somewhat lower in the liver samples taken 4-10 min. after decapitation. Experiments with DPN added to liver in amounts approximately equal to those found in the tissues resulted in a 91% recovery at pH 7.4. The recovery of added DPNH at pH 7.4 was poor (30-7OoJo). At pH 9.3, however, in the presence of lOO200 mg. liver, recovery of added DPNH was excellent (102yo). Since the endogenous DPNH concentrations were the same at pH’s 7.4, 9.3, and 11.0, and at different tissue levels, it was obvious that the extraction of DPN and DPNH could be performed simultaneously at pH 7.4. Apparently, under the conditions used here, exogenous, but not endogenous, DPNH is partly destroyed between pH 7.4 and 9.3. This method is being extended to other tissues and animals under a variety of physiological and pathological conditions. REFERENCES
1. EULER, H. VON, Ergeb. Physiol., biol. Chem. u. ezptl. Pha~mak01. 38, 1 (1936). 2. FISHER, A., AND SCHLENK, F., Texus Repts. Biol. and Med. 6, 346 (1948). 3. BERNHEIM, F., AND FELSOVANYI, A. VON, Science 91, 76 (1940). 4. JANDORF, B. J., J. Biol. Chem. 159, 89 (1943). 5. AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol. Chem. 137,77 (1939). 6. KENSLER, C. J., SUQIURA, K., AND RHOADS, C. P., Science 91. 623, (1940).
LETTERS
TO
THE
311
EDITORS
7. HOBECHEB, B. A., AND KORNBEBG, A., J. Biol. Chem. 176,386 (1948). 8. RACKER, E., J. Biol. Chem. 184,313 (1950). 9. STRENGTH, D. R., RINGLER, I., AND NELSON, W. L., Arch. Biochem. and Biophys. 48, 107 (1954). 10. HOLZER, H., GOLDSCHMIDT, S., LAMPRECHT, W., AND HELMREICH, E., 2. physiol. Chem. !W7, 1 (1954). 11. JEDEIKIN, L., AND WEINHOUSE, S., as quoted by GREENSTEIN, J. P., in “Bio-
chemistry 1954. 12. STRENGTH, Biophys.
13. GUTCHO,
of Cancer,“2nd
ed., pp. 371-2. Academic Press, New York, N. Y.,
D. R., MONDY, 48, 141 (1954). S., AND STEWART,
N. I., AND DANIEL, E. D., Anal.
of Pharmacology and William Goldman Isotope Laborato Division of Biological Chemistry, Hahnemunn Medical College, Philadelphia, Pennsylvania Received September 4, 1964
Chem.
L. J., Arch.
Observations
(1948). M. A. SPIBTES HERBERT J. EICHEL
of EDTA
with the Myosin-ATP
y,
on the Interaction System
and
20,185
Division
Some Further
Biochem.
In previous papers (1, 2) it was reported that ethylenediaminetetraacetic acid (EDTA) was a powerful activator of the myosin-adenosine triphosphate (ATP) system in 0.6 M KC1 at pH 7.0. It was suggested (1) that in this effect EDTA was either acting directly on the myosin (an improbability but not an impossibility) or else through the intervention of an alkaline-earth cation. In the present communication we wish to report recent results which further narrow the range of explanations. Reaction rates were measured in 0.6 A4 KCl, pH 7, at 25°C.; all techniques and materials have been described in an earlier paper (1). Mg has been throughout a likely candidate for the intermediary in the action of EDTA: the known effect of Mg++ on adenosinetriphosphatase (ATPase) activity, acceleration at low and inhibition at high concentrations of KC1 (3), fits in well with the effects of EDTA in the corresponding milieus (1,2); chelating agents other than EDTA which mimic the EDTA effect are the agents known to chelate Mg++ (2); Mg-EDTA has no accelerating effect upon ATPase (2); by direct analysis’ myosin contains roughly 0.1 pg./mg. (range of nine measurements on five different protein preparations (5): 0.03-0.18) of alkaline-earth metals (mostly Mg).* However, the facts that myosin exhaustively dialyzed against EDTA, then washed free of EDTA, will respond to a second addition of EDTA (I), and that it retains its Mg content unchanged, argue against the notion that Mg++ existed in the untreated system as a relatively mobile impurity. Two additional experiments assure this conclusion. First we measured the dependence of maximum 1 W. J. Bowen and T. D. Kerwin, unpublished data. 2 In these experiments, of course, the myosin was prewashed KC1 solution.
with
Mg-free
LETTERS
TO
THE
EDITORS
basis of ultraviolet spectrophotometry, 1.4 mg. was isolated, which represent 0.5oJc of the administered cortisol. By a similar procedure, late human pregnancy urine (a a-day urine collection) was extracted and the extract chromatographed on paper. The most polar reducing zone was acetylated and rechromatographed on paper. Several cr,fl-unsaturated ketonic steroids were observed under ultraviolet light. The major reducing zone was eluted. On the basis of ultraviolet spectrophotometry, 300 pg. of material was present. The material could not be crystallized, but the infrared analysis and the absorption spectrum in sulfuric acid indicated that 6p-hydroxycortisol was present. This is the first instance of in. &JO 6&hydroxylation in the human. Further studies on the excretion of G&hydroxycortisol in various pathological and stress conditions are being done to establish the biological significance of the 6&hydroxylation in vivo. REFERENCES 1. BURSTEIN,
S., DORFMAN,
R. I., AND NADEL,
E. M., Federation
Proc. 13, 188
(1954). 2. LIEBERMAN,
S., FUKUSHIMA,
D. K., AND DOBRINER,
K., J. BioZ. Chem. 182,299
(1950). 3. BURSTEIN,
S., AND DORFMAN,
R. I., J. Biol. Chem. !XMf, 607 (1954).
Worcester Foundation for Experimental Biology, Shrewsbuy, Massachusetts National Cancer Institute, National Institutes of Health, U. S. Department of Health, Education and Welfare, Bethesda, Maryland Received September dS,196+$ A Single-Extract
SHLOMO BURSTEIN RALPH I. DORFMAN ELI M. NADEL
Method for the Determination of Oxidized Diphosphopyridine Nucleotide in Rat Liver’
and Reduced
The three methods used most in the past for the determination of DPNz and DPNH in tissues are cumbersome and dependent upon the simultaneous standardization and assay of purity of a DPN preparation (l-4). With these three techniques, values of total rat liver DPN ranging from 500 to 1400 pg./g. fresh weight have been reported (2-6). It is possible that the higher figures are a reflection of the use of DPN standards the purities of which were lower than supposed.3 Since highly purified DPN preparations with known molar extinction co1 This investigation was supported in part by a grant from the U. S. Atomic Energy Commission (Contract No. AT(30-l)-1069) and a grant (A-530) from the National Institutes of Health, U. S. Public Health Service. 2 The following abbreviations are employed: DPN, diphosphopyridine nucleotide; DPNH, dihydrodiphosphopyridine nucleotide; ADH, alcohol dehydrogenase. * Dr. F. Schlenk, personal communication.
LETTERS
TO
THE
EDITORS
309
efficients (7) have been available for some time and a simple, one-enzyme method for the determination of both oxidized and reduced DPN has been described (8), it was decided to apply these findings to reinvestigate the levels of DPN and ‘DPNH in rat liver. Two recently published procedures for the analysis of tissue DPN and DPNH also take advantage of these findings. Thus, Strength et al. (9) reported a method for the determination of rat liver DPN based on extraction with trichloroacetic acid and assay with either lactic dehydrogenase or ADH. Holzer et. al. (10) analyzed rat liver and yeast for their content of DPN and DPNH by extraction with acid and alkali, respectively, and assay with ADH. In the present communication, conditions are described for the simultaneous extraction of both DPN forms from one liver sample. Levels of total liver DPN obtained by this technique average 863 pg./g. fresh weight, a value in good agreement with the data of Jandorf (4) and higher than many figures quoted in the literature (2-6, 10-12). Into each of two Potter-Elvehjem homogenizers are placed 2 ml. of 0.1 M KHzPOl , 2 ml. of a solution containing 0.5% NaHCOr0.5% NazCOa , and 3 ml. of glass-distilled water. The homogenizers are placed in a boiling water bath at 2-min. intervals. Two minutes after inserting the first homogenizer into the water bath, a rat is decapitated and the liver removed immediately and blotted free of blood. A weighed portion of 400-500 mg. of liver (kept at room temperature) is added to the first homogenizer 34 min. after it has been in the bath. After a half minute, the homogenizer is removed, and the liver is homogenized for 30 sec. The homogenizer is then replaced in the water bath for an additional half minute and finally plunged into a container of ice. Another portion of liver is now weighed and added to the second homogenizer 3 min. after it has been in the bath, and the same process is repeated. Both chilled homogenates are diluted quantitatively to 15 ml. with glass-distilled water and centrifuged at 34,850 X 9 for 13 min. in a Spinco ultracentrifuge (model L, rotor No. 30). The yellowish, almost clear supernatants are removed and added to previously chilled test tubes, and the latter are kept cold until used. DPN and DPNH determinations are performed according to Racker (8). One and a half milliliters of extract in a total volume of 3 ml. was found to give good readings with the Beckman DU spectrophotometer at 340 rnp. ADH was obtained from Worthington Biochemical Co. as a suspension in ammonium sulfate; 0.03 ml. of a 1:50 dilution of the enzyme was sufficient to drive both reactions to completion within several minutes. DPN and DPNH were obtained from Sigma. DPNH samples were also prepared chemically (13) and enzymatically with ADH and used without further purification. For a typical DPN determination, the following components are added to the cuvette in order: 1.5 ml. liver extract, 0.3 ml. of 0.1 M pyrophosphate buffer, pH 9.0, 0.1 ml. of 95% ethanol, and water to make a volume of 2.97 ml. After mixing, duplicate determinations of the optical density are made. Following the addition of 0.03 ml. of ADH and careful mixing, the density is recorded at l-mm. intervals until at least two or three unchanged readings are obtained, at which point the reaction is considered complete. For the determination of DPNH, a similar procedure is followed except that 0.3 ml. of 0.1 M KzHPO,-KHzPOI buffer, pH 7.1, is substituted for the pyrophosphate, and 0.1 ml. of 10% redistilled acetaldehyde for the ethanol. The absorption coefficient of 6.22 X lo6 sq. cm./ mole, derived by Horecker and Kornberg (7) for the 340-m& band of DPNH, is used to calculate
310
LETTERS
TO
THE
EDITORS
TABLE Concentration
of Oxidized
and Reduced
I Diphosphopyridine
Nueleotide
in
DPN /DPNH
<2.5
8
4-10
8
51gb (488-552) 415 (30849)
345 (321-418) 338 (308-411)
(79212) 753 (635-867)
(l.lZ2.74) 1.25 (0.94-1.76)
a Wistar males, 110-159 g. b The values represent the average and range. the results. The final reading minus the initial reading, in the case of DPN, and the initial reading minus the final reading, in the case of DPNH, are used to calculate the levels of the two forms of the coenzyme present in the extract. Table I lists liver DPN, DPNH, and DPN/DPNH values of normal male Wistar rats (110-150 g.). It can be seen that the average total figure of 863 ag./g. fresh weight obtained for liver samples placed into the hot medium 2.5 min. or less after decapitation was consistently higher than the mean of 751 pg./g. found for samples taken 4-10 min. after decapitation. This observation corroborates Jandorf’s (4) earlier data. Table I shows that the decline in total coenzyme concentration is due to a decrease in oxidized component. No change occurs in the DPNH concentration during the lo-min. period following decapitation. Accordingly, the DPN/DPNH ratios are somewhat lower in the liver samples taken 4-10 min. after decapitation. Experiments with DPN added to liver in amounts approximately equal to those found in the tissues resulted in a 91% recovery at pH 7.4. The recovery of added DPNH at pH 7.4 was poor (30-7OoJo). At pH 9.3, however, in the presence of lOO200 mg. liver, recovery of added DPNH was excellent (102yo). Since the endogenous DPNH concentrations were the same at pH’s 7.4, 9.3, and 11.0, and at different tissue levels, it was obvious that the extraction of DPN and DPNH could be performed simultaneously at pH 7.4. Apparently, under the conditions used here, exogenous, but not endogenous, DPNH is partly destroyed between pH 7.4 and 9.3. This method is being extended to other tissues and animals under a variety of physiological and pathological conditions. REFERENCES
1. EULER, H. VON, Ergeb. Physiol., biol. Chem. u. ezptl. Pha~mak01. 38, 1 (1936). 2. FISHER, A., AND SCHLENK, F., Texus Repts. Biol. and Med. 6, 346 (1948). 3. BERNHEIM, F., AND FELSOVANYI, A. VON, Science 91, 76 (1940). 4. JANDORF, B. J., J. Biol. Chem. 159, 89 (1943). 5. AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol. Chem. 137,77 (1939). 6. KENSLER, C. J., SUQIURA, K., AND RHOADS, C. P., Science 91. 623, (1940).
LETTERS
TO
THE
311
EDITORS
7. HOBECHEB, B. A., AND KORNBEBG, A., J. Biol. Chem. 176,386 (1948). 8. RACKER, E., J. Biol. Chem. 184,313 (1950). 9. STRENGTH, D. R., RINGLER, I., AND NELSON, W. L., Arch. Biochem. and Biophys. 48, 107 (1954). 10. HOLZER, H., GOLDSCHMIDT, S., LAMPRECHT, W., AND HELMREICH, E., 2. physiol. Chem. !W7, 1 (1954). 11. JEDEIKIN, L., AND WEINHOUSE, S., as quoted by GREENSTEIN, J. P., in “Bio-
chemistry 1954. 12. STRENGTH, Biophys.
13. GUTCHO,
of Cancer,“2nd
ed., pp. 371-2. Academic Press, New York, N. Y.,
D. R., MONDY, 48, 141 (1954). S., AND STEWART,
N. I., AND DANIEL, E. D., Anal.
of Pharmacology and William Goldman Isotope Laborato Division of Biological Chemistry, Hahnemunn Medical College, Philadelphia, Pennsylvania Received September 4, 1964
Chem.
L. J., Arch.
Observations
(1948). M. A. SPIBTES HERBERT J. EICHEL
of EDTA
with the Myosin-ATP
y,
on the Interaction System
and
20,185
Division
Some Further
Biochem.
In previous papers (1, 2) it was reported that ethylenediaminetetraacetic acid (EDTA) was a powerful activator of the myosin-adenosine triphosphate (ATP) system in 0.6 M KC1 at pH 7.0. It was suggested (1) that in this effect EDTA was either acting directly on the myosin (an improbability but not an impossibility) or else through the intervention of an alkaline-earth cation. In the present communication we wish to report recent results which further narrow the range of explanations. Reaction rates were measured in 0.6 A4 KCl, pH 7, at 25°C.; all techniques and materials have been described in an earlier paper (1). Mg has been throughout a likely candidate for the intermediary in the action of EDTA: the known effect of Mg++ on adenosinetriphosphatase (ATPase) activity, acceleration at low and inhibition at high concentrations of KC1 (3), fits in well with the effects of EDTA in the corresponding milieus (1,2); chelating agents other than EDTA which mimic the EDTA effect are the agents known to chelate Mg++ (2); Mg-EDTA has no accelerating effect upon ATPase (2); by direct analysis’ myosin contains roughly 0.1 pg./mg. (range of nine measurements on five different protein preparations (5): 0.03-0.18) of alkaline-earth metals (mostly Mg).* However, the facts that myosin exhaustively dialyzed against EDTA, then washed free of EDTA, will respond to a second addition of EDTA (I), and that it retains its Mg content unchanged, argue against the notion that Mg++ existed in the untreated system as a relatively mobile impurity. Two additional experiments assure this conclusion. First we measured the dependence of maximum 1 W. J. Bowen and T. D. Kerwin, unpublished data. 2 In these experiments, of course, the myosin was prewashed KC1 solution.
with
Mg-free