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Effect of methylene blue on the metabolism of adenine nucleotldes in human erythrocytes
580
LETTERS
4. LORING,
5. DRURY,
TO
THE
H. S., LEVY, L. W., AND Moss, H. F., Arch. Biochem. 19, 455
EDITORS
L. K.,
Anal.
Department of Chemistry and School of Medicine, Stanford University, Stanford, California Received October 17, 1966
Effect of Methylene
Chem. 28, 539 (1956).
(1948). HUBERT S. LORINC LLOYD K. Moss LUIS W. LEVY WM. F. HAIN
Blue on the Metabolism of Adenine Nucleotides in Human Erythrocytesl
Since the publieation of the observations of Guzman Barron and others (1) it has been known that the rate of uptake of oxygen in erythrocytes is enhanced in the presence of methylene blue. These and some further studies (2) led to the discovery of the nonglycolytic mechanism of glucose metabolism in these cells. It was found that the nonglycolytic “pathway” was stimulated by the presence of methylene blue. The addition of substances connected with the tricarboxylic cycle to the suspension medium of red cells has been described to provide “substrate” for the effect of methylene blue on oxygen uptake (3). Dimercaprol (BAL) has also been described as a substance capable of increasing the rate of oxygen uptake in erythrocyt,es, and also in this case, a mechanism involving the direct oxidation of glucose-g-phosphate was suggested (4). We have been interested in the question, whether methylene blue and dimercaprol would show any demonstrable effect on the turnover rates of phosphorus in the adenosine nucleotides of human red cells. Our interest was partly derived from our earlier observations on certain differences in the metabolism of phosphorus compounds in erythrocytes of patients suffering from schizophrenia (5, 6). Blood was drawn from the cubital vein of fasting subjects. The latter were females in their earliest twenties (student nurses). Heparin was used as anticoagulant. After having been stored over ice for a period of not longer than 2 hr., the blood samples were incubated with constant shaking in open flasks for 60 min. Shortly before the beginning of the incubation Sodium-Radio-Phosphate (Abbott) was added in a concentration of 20 pc./lO ml. of blood. At the end of the incubation the blood samples were chilled, the plasma and the layer of white cells discarded after centrifugation, and the erythrocyte cream was extracted with cold trichloroacetic acid (final concentration: 770 w/v). A barium-alcohol precipitation was done (7) at pH 8.2 and, after redissolving in HCl, barium was removed as sulfate. After vacuum concentration the samples were applied on Munktell No. 20s paper, soaked in citrate buffer (pH 3.9). Electrophoresis in an LKB apparatus gave clear separation of several ultraviolet-absorbing spots, among which ATP and ADP were easily recognizable and identified. The ratio of the counts per minute value of the individual spot to the corresponding As260--290mp readings in a Beckmann spectrophotometer was taken as the relative specific activity of the nucleotide concerned. 1 Supported by funds from the Scottish-Rite University of Illinois Foundation.
Research
Committee,
and by the
LETTERS
TO
THE
TABLE Relative
EDITORS
581
I
Specific Activity Values of the ADP Spots from Aliquots of the Same Blood Sample under Various Conditions of Incubation
No. of expts.
“Blank”
3
1.53
Pyruvate and citrate 5 mg. of Na salts in 10 ml. of blood
Methylene blue plus pynlvate and citrate
1.40
1.95
1.71 TABLE
II
Comparative Effects of Methylene Blue (M.B.) on the Relative Speci$c Activities the ADP and ATP Spots in the Presence of Pyruvate and Citrate (6 mg. of the Na Salts of Each in 10 ml. of Blood) ADP No. of expts.
No hf. B.
29
1.46
2.5
of
ATP Per cent increase with
Per cent
x”io”h Y
“pn from ,y>
NoM.B.
1.97
+35.20/o
2.88
M. B.
2.59
<0.091
decrease up,, fromwith ,y,
-10.1%
As it is apparent from data in Table I, the presence of methylene blue (2.5 X 10-K M) increased the rate of incorporation of Paz in the ADP molecule, and especially so in the presence of citrate and pyruvate additions (5 mg. of the sodium salt of each in 10 ml. of blood). In Table II the effect of methylene blue in the presence of the latter additions is compared as it affected the relative P32 specific activities in the ADP and ATP fractions of a larger number of samples from healthy donors. The turnover rate of Pa* in ATP was decreased by methylene blue. Dimercaprol had an effect similar to methylene blue on ADP but no effect on the turnover rate of ATP (Table III). We have not found an explanation for the above phenomena on the basis of known metabolic processes described in erythrocytes. It is remarkable that an increase in the turnover rate of phosphorus in ADP appears to be associated with a decrease of the rate in the ATP fraction. It is also to be remembered that the effect of methylene blue on the uptake of oxygen is of a higher percentile order than the effect described here. Whereas a decrease in the uptake of Pa2 in ATP could be due to the known “uncoupling” effect of methylene blue, such effect could hardly account for an actual increase of the relative specific activity of the ADP. A reaction involving nucleotides: the synthesis of nicotinamide monoTABLE
III
Effect of Dimercaprol (BAL in Oil lo%, 0.03 ml. to 10 ml. of Blood) on the Relative Specijic Activity Values of ADP and ATP in the Presence of Pyruvate and Citrate (6 mg. of the Na Salts of Each in 10 ml. of Blood) ATP
ADP No. of expts.
No BAL
BAL
21
1.42
1.90
Per cent increase with ‘,P” from 0,”
+33.8
No BAL
2.90
BAL
2.84
Per cent decrease
-2.2
LETTERS
582
TO
nucleotide was reported to be unaffected suspensions of human erythrocytes (8).
THE
EDITORS
by the presence
of methylene
blue in
REFERENCES 1. 2. 3. 4. 5.
HARROP, A., AND BARRON, E. S. G., J. Esptl. Med. 48, 207 (1928). WARBURG, O., AND CHRISTIAN, W., B&hem. 2. 343, 206 (1931). NOSSAL, P. M., Australian J. Exptl. Biol. Med. Sci. 26, 123 (1948). REPKE, K., Arch. Exptl. Pathol. Pharmakol. 224, 300 (1955). BOSZORMENYI-NAGY, I., AND GERTY, F. J., J. Nervous Mental Disease 121, 53 (1955). 6. BOSZORMENYI-NAGY, I., ASD GERTY, F. J., Am. J. Psych&. 112, 11 (1955). 7. LEPAGE, G. A., in “Manometric Methods and Tissue Metabolism” (Umbreit, W. W., Burris, R. H., and Stauffer, J. F., eds.) p. 185, Burgess Publ. Co., Minneapolis, 1949. 8. LEDER, I. G., AND HANDLER, P., J. Biol. Chem. 182, 889 (1951). Division of Psychiatry, Neuropsychiatric Institute, of Illinois, University College of Medicine, Chicago, Illinois Received September lY, 1956
Conversion
IVAN BOSZORMENYI-NAGY DORIS BLACKFORD
of Argininosuccinic Acid to Citrulline Coupled to ATP Formation’
Earlier investigations have shown that ASA,e the immediate precursor of arginine, is formed by an ATP-utilizing condensation of citrulline with aspartic acid (1, 2). Two enzymes participate in promoting the condensation, both originally present in liver. Enzyme A was prepared from mammalian liver and Erlzyme B from yeast. The activity of each purified enzyme was assayed by providing an excess of the other. Their respective roles in the reaction mechanism and the stoichiometry of the phosphate transfer were uncertain (3). It now appears that Enzyme B is identical with the enzyme PPase. Evidence for this was obtained by comparing PPase activity with Enzyme B activity during the preparation of crystalline PPase (Table I). The ratio of the two activities remained substantially the same throughout fractionation. With PP thus implicated in the condensation mechanism, it would appear that the cleavage of PP from ATP accompanies the condensation catalyzed by Enzyme A, and that Enzyme B (PPase) exerts its strong acceleration (four to fivefold) by 1 Aided by a Grant from the American Cancer Society. 2 Abbreviations: argininosuccinic acid, ASA; adenosine mono-, di-, and triphosphate, AMP, ADP, and ATP; inorganic pyrophosphate, PP; inorganic pyrophosphatase, PPase; orthophosphate, Pi; glucose-6.phosphate, G-6-p; trichloroacetic acid, TCA.
LETTERS
4. LORING,
5. DRURY,
TO
THE
H. S., LEVY, L. W., AND Moss, H. F., Arch. Biochem. 19, 455
EDITORS
L. K.,
Anal.
Department of Chemistry and School of Medicine, Stanford University, Stanford, California Received October 17, 1966
Effect of Methylene
Chem. 28, 539 (1956).
(1948). HUBERT S. LORINC LLOYD K. Moss LUIS W. LEVY WM. F. HAIN
Blue on the Metabolism of Adenine Nucleotides in Human Erythrocytesl
Since the publieation of the observations of Guzman Barron and others (1) it has been known that the rate of uptake of oxygen in erythrocytes is enhanced in the presence of methylene blue. These and some further studies (2) led to the discovery of the nonglycolytic mechanism of glucose metabolism in these cells. It was found that the nonglycolytic “pathway” was stimulated by the presence of methylene blue. The addition of substances connected with the tricarboxylic cycle to the suspension medium of red cells has been described to provide “substrate” for the effect of methylene blue on oxygen uptake (3). Dimercaprol (BAL) has also been described as a substance capable of increasing the rate of oxygen uptake in erythrocyt,es, and also in this case, a mechanism involving the direct oxidation of glucose-g-phosphate was suggested (4). We have been interested in the question, whether methylene blue and dimercaprol would show any demonstrable effect on the turnover rates of phosphorus in the adenosine nucleotides of human red cells. Our interest was partly derived from our earlier observations on certain differences in the metabolism of phosphorus compounds in erythrocytes of patients suffering from schizophrenia (5, 6). Blood was drawn from the cubital vein of fasting subjects. The latter were females in their earliest twenties (student nurses). Heparin was used as anticoagulant. After having been stored over ice for a period of not longer than 2 hr., the blood samples were incubated with constant shaking in open flasks for 60 min. Shortly before the beginning of the incubation Sodium-Radio-Phosphate (Abbott) was added in a concentration of 20 pc./lO ml. of blood. At the end of the incubation the blood samples were chilled, the plasma and the layer of white cells discarded after centrifugation, and the erythrocyte cream was extracted with cold trichloroacetic acid (final concentration: 770 w/v). A barium-alcohol precipitation was done (7) at pH 8.2 and, after redissolving in HCl, barium was removed as sulfate. After vacuum concentration the samples were applied on Munktell No. 20s paper, soaked in citrate buffer (pH 3.9). Electrophoresis in an LKB apparatus gave clear separation of several ultraviolet-absorbing spots, among which ATP and ADP were easily recognizable and identified. The ratio of the counts per minute value of the individual spot to the corresponding As260--290mp readings in a Beckmann spectrophotometer was taken as the relative specific activity of the nucleotide concerned. 1 Supported by funds from the Scottish-Rite University of Illinois Foundation.
Research
Committee,
and by the
LETTERS
TO
THE
TABLE Relative
EDITORS
581
I
Specific Activity Values of the ADP Spots from Aliquots of the Same Blood Sample under Various Conditions of Incubation
No. of expts.
“Blank”
3
1.53
Pyruvate and citrate 5 mg. of Na salts in 10 ml. of blood
Methylene blue plus pynlvate and citrate
1.40
1.95
1.71 TABLE
II
Comparative Effects of Methylene Blue (M.B.) on the Relative Speci$c Activities the ADP and ATP Spots in the Presence of Pyruvate and Citrate (6 mg. of the Na Salts of Each in 10 ml. of Blood) ADP No. of expts.
No hf. B.
29
1.46
2.5
of
ATP Per cent increase with
Per cent
x”io”h Y
“pn from ,y>
NoM.B.
1.97
+35.20/o
2.88
M. B.
2.59
<0.091
decrease up,, fromwith ,y,
-10.1%
As it is apparent from data in Table I, the presence of methylene blue (2.5 X 10-K M) increased the rate of incorporation of Paz in the ADP molecule, and especially so in the presence of citrate and pyruvate additions (5 mg. of the sodium salt of each in 10 ml. of blood). In Table II the effect of methylene blue in the presence of the latter additions is compared as it affected the relative P32 specific activities in the ADP and ATP fractions of a larger number of samples from healthy donors. The turnover rate of Pa* in ATP was decreased by methylene blue. Dimercaprol had an effect similar to methylene blue on ADP but no effect on the turnover rate of ATP (Table III). We have not found an explanation for the above phenomena on the basis of known metabolic processes described in erythrocytes. It is remarkable that an increase in the turnover rate of phosphorus in ADP appears to be associated with a decrease of the rate in the ATP fraction. It is also to be remembered that the effect of methylene blue on the uptake of oxygen is of a higher percentile order than the effect described here. Whereas a decrease in the uptake of Pa2 in ATP could be due to the known “uncoupling” effect of methylene blue, such effect could hardly account for an actual increase of the relative specific activity of the ADP. A reaction involving nucleotides: the synthesis of nicotinamide monoTABLE
III
Effect of Dimercaprol (BAL in Oil lo%, 0.03 ml. to 10 ml. of Blood) on the Relative Specijic Activity Values of ADP and ATP in the Presence of Pyruvate and Citrate (6 mg. of the Na Salts of Each in 10 ml. of Blood) ATP
ADP No. of expts.
No BAL
BAL
21
1.42
1.90
Per cent increase with ‘,P” from 0,”
+33.8
No BAL
2.90
BAL
2.84
Per cent decrease
-2.2
LETTERS
582
TO
nucleotide was reported to be unaffected suspensions of human erythrocytes (8).
THE
EDITORS
by the presence
of methylene
blue in
REFERENCES 1. 2. 3. 4. 5.
HARROP, A., AND BARRON, E. S. G., J. Esptl. Med. 48, 207 (1928). WARBURG, O., AND CHRISTIAN, W., B&hem. 2. 343, 206 (1931). NOSSAL, P. M., Australian J. Exptl. Biol. Med. Sci. 26, 123 (1948). REPKE, K., Arch. Exptl. Pathol. Pharmakol. 224, 300 (1955). BOSZORMENYI-NAGY, I., AND GERTY, F. J., J. Nervous Mental Disease 121, 53 (1955). 6. BOSZORMENYI-NAGY, I., ASD GERTY, F. J., Am. J. Psych&. 112, 11 (1955). 7. LEPAGE, G. A., in “Manometric Methods and Tissue Metabolism” (Umbreit, W. W., Burris, R. H., and Stauffer, J. F., eds.) p. 185, Burgess Publ. Co., Minneapolis, 1949. 8. LEDER, I. G., AND HANDLER, P., J. Biol. Chem. 182, 889 (1951). Division of Psychiatry, Neuropsychiatric Institute, of Illinois, University College of Medicine, Chicago, Illinois Received September lY, 1956
Conversion
IVAN BOSZORMENYI-NAGY DORIS BLACKFORD
of Argininosuccinic Acid to Citrulline Coupled to ATP Formation’
Earlier investigations have shown that ASA,e the immediate precursor of arginine, is formed by an ATP-utilizing condensation of citrulline with aspartic acid (1, 2). Two enzymes participate in promoting the condensation, both originally present in liver. Enzyme A was prepared from mammalian liver and Erlzyme B from yeast. The activity of each purified enzyme was assayed by providing an excess of the other. Their respective roles in the reaction mechanism and the stoichiometry of the phosphate transfer were uncertain (3). It now appears that Enzyme B is identical with the enzyme PPase. Evidence for this was obtained by comparing PPase activity with Enzyme B activity during the preparation of crystalline PPase (Table I). The ratio of the two activities remained substantially the same throughout fractionation. With PP thus implicated in the condensation mechanism, it would appear that the cleavage of PP from ATP accompanies the condensation catalyzed by Enzyme A, and that Enzyme B (PPase) exerts its strong acceleration (four to fivefold) by 1 Aided by a Grant from the American Cancer Society. 2 Abbreviations: argininosuccinic acid, ASA; adenosine mono-, di-, and triphosphate, AMP, ADP, and ATP; inorganic pyrophosphate, PP; inorganic pyrophosphatase, PPase; orthophosphate, Pi; glucose-6.phosphate, G-6-p; trichloroacetic acid, TCA.