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Synthesis of pantothenic acid by depyrophosphorylation of adenosine triphosphate
236
LETTERS A Spray for the Differentiation
TO THE EDITORS
Between Aureomycin Chromatogramsi
and Terramydn
on
One of the major problems in screening for new antibiotics is the rapid recognition of known active substances. A spray consisting of a 2% solution (w/v) of p-dimethylaminobenzaldehyde (PDAB) in 1.2 N hydrochloric acid and employed in the identification of allantoin and urea (1) also has proven useful in locating aureomycin and terramycin on paper chromatograms and in differentiating between these two antibiotics. After 5-8 hr. at room temperature, aureomycin shows ,up as a dirty yellow spot while terramycin gives a blue-green color. Approximately 5 pg. of the antibiotics applied over an area of 1 cm. in diameter is the smallest amount that can be detected. Chloromycetin, in quantities greater than 5 pg., can also be detected with this reagent, if the chromatogram is first sprayed with a 0.25% solution of stannous chloride in 1 N hydrochloric acid and allowed to dry. This procedure is a slight modification of the method of Smith and Worrel (2). After spraying with the PDAB reagent, a bright yellow spot appears after 5 min. at, room temperature. Qualitative reactions like these should increase the confidence with which the tentative identification of antibiotics on the basis of RI values in various solvent systems is regarded. REFERENCES 1. BERRY, H. K., SUTTON, H. E., CAIN, L., AND BERRY, J. S., Univ. Texas Publ. (Biochemical Institute Studies), No. IV (1951). 2. SMITH, G. N., AND WORREL, C. S., Arch. Biochem. 28, 1 (1959). W. T. SOKOLSKI HENRY KOFFLER P. A. TETRAULT
Laboratories Department
of Bacteriology, of Biological Sciences, Purdue University, Lafayette, Indiana Received December 1, 1969
Synthesis of Pantothenic Acid by Depyrophosphorylation Adenosine Triphosphate
of
Lipmann et al. (1) have recently reported that, in the enzymatic synthesis of acetyl-CoA, the energy transfer is effected by a pyrophosphate-liberating split of adenosine triphosphate (ATP). The synthesis of pantothenic acid, which previously was shown to proceed with ATP as energy source (2), now seems to involve a similar mechanism, evidenced by the appearance of inorganic pyrophosphate (PPi) and adenylic acid (AMP) as end products. This finding indicates a more general occurrence of depyrophosphorylation in energy transfer, especially since the synt.hesis of pantothenic acid does not seem to involve coenzyme A (CoA) (2). An experiment demonstrating the balance of reactants and products is shown in Table I. The enzyme used for this experiment was extrscted.from acetone1 Supported
in part by a grant from the Squibb Institute
for Medical Research.
LETTERS
TO
THE
237
EDITORS
dried cells of E’scherichia coli as described previously (2) and freed from most of the contaminating adenosinetriphosphatase (ATPase), myokinase, and pyrophosphatase by protamine and ammonium sulfate precipitations. Pantothenate was determined by microbiological assay with a mutant of E. coli (3); AMP spectrophotometrically according to Kalckar (4) using Schmidt’s deaminase; pyrophosphate as easily hydrolyzable phosphate after removal of ATP with charcoal (Norit) (5) ; ATP by difference in easily hydrolyzable phosphate before and after charcoal adsorption; inorganic orthophosphate (Pi) by the FiskeSubbaRow method (6). The identity and amount of pyrophosphate was checked further by treatment with five times recrystallized pyrophosphatase (7), kindly supplied by Dr. Kunitz, by acid hydrolysis of the Mn salt (8), and by paper chromatography (9); pantothenate, AMP, and ATP were also identified by paper chromatography in several solvent systems. TABLE
I
Balance of Reactants and Products Each tube received: 40 rmoles &alanine, 40 pmoles potassium pantoate, 40 pmoles ATP, 200 pmoles KCl, 40 pmoles MgClr, 200 pmoles KHCOI (pH 8.1), in a total volume of 2.0 ml. To each vessel, 0.1 ml. of extract containing 0.72 mg. protein was added. Incubation was at 37” for 80 min. At the beginning and the end of incubation samples were removed, inactivated by heating or treatment with trichloroacetic acid, and analyzed. Reaction mixture
Initial Final
Pantothenate pmoler/ml.
0.05 9.4
AMP pmolcs/ml.
@tOlES/9d.
PPi
0.3 9.5
0 7.2
Pi
ATP
@&.r/?nl.
pWlCS/d.
1.2 2.2
19.9 11.2
These data show a stoichiometric relationship between the amount of pantothenate synthesized and that of ATP split into AMP and pyrophosphate, suggesting the following over-all reaction : &alanine
+ pantoic
acid + ATP -+ pantothenic
acid + AMP + PPi
In support of the specific nature of the splitting of ATP, it was found that adenosine diphosphate (ADP) does not substitute for ATP as energy source. REFERENCES
F., JONES, M. E., BLACK, S., AND FLYNN, R. M., J. Am. Chem. 74, 2384 (1952). MAAS, W. K., J. Biol. Chem. 198,23 (1952). MAAS, W. K., AND DAVIS, B. D., J. Bact. 60, 733 (1950). KALCKAR, H. M., J. Biol. Chem. 167,445 (1947). CRANE, R. K., AND LIPMANN, F., J. Biol. Chem., in press. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 68,375 (1925).
1. LIPMANN,
2. 3. 4. 5. 6.
SOC.
238
LETTERS
7.
KUNITZ,
8. 9.
K?RNB.ERG, A., J. Biol. EBEL, J., AND VOLMAR,
THE
EDITORS
M., J. Gen. Physiol. 36, 423 (1962). Chem. 162, 779 (1950). Y., Compt. rend. 233,416
Biochemical Research Massachusetts General logical
TO
(1951).
Laboratory, Hospital
WERNER
and the Department
of Bio-
K.
G. DAVID
MAAS’ NOVELLI
Chemistry,
Harvard Medical School, Boston, Received January 21, 1965
Massachusetts
Paper Chromatography
of Some Ketoheptosesl
Klevstrand and Nordal (1) indicated that a spray reagent containing orcinol and trichloroacetic acid in water-saturated butanol was apparently specific for identifying ketoheptoses (sedoheptulose and mannoheptulose) on paper chromatograms. Bevenue and Williams (2) confirmed this observation with D-glucoTABLE Paper Chromatographic Sugar
n-Glucoheptulose n-Mannoheptulose n-Galaheptulose Sedoheptulose n-Idoheptulose L-Guloheptulose Sedoheptulosan . n-Idoheptulosan L-Guloheptulosan
Behavior RI (phenol)
0.42 0.40 0.48 0.46 0.60 0.53 0.70 0.64 0.65
I of Some
Ketoheptoses
Distance from origin (ethyl acetate) nm.
103 108 99 125 147 138 153 194 188
Color
with spray
reagent
Blue Green-blue Blue Blue Blue Blue Blue Blue Blue
heptulose in addition to sedoheptulose and mannoheptulose but further stated that sedoheptulosan did not give a color with the spray reagent. This latter observation was not in agreement with results on sedoheptulosan samples run in this laboratory, so some additional work was attempted. Through the courtesy of Dr. Nelson K. Richtmyer of the National Institutes of Health, Bethesda, Maryland, a number of ketoheptose samples were obtained, which included n-glucoheptulose, n-mannoheptulose, L-galaheptulose, sedoheptulosan monohydrate, n-idoheptulosan, and L-guloheptulosan hemihydrate. The last three samples were anhydrides, and to obtain a mixture of the free sugar and anhydride the anhydrides were treated with hot 0.2 N HCl as was described for the preparation of n-idoheptulose and n-idoheptulosan (3). The free sugars and anhydrides 1 Senior Asst. Scientist, U. S. Public Health Service. 1 Work performed under Contract No. W-7406-eng-26 Energy Commission.
for the U. S. Atomic
LETTERS A Spray for the Differentiation
TO THE EDITORS
Between Aureomycin Chromatogramsi
and Terramydn
on
One of the major problems in screening for new antibiotics is the rapid recognition of known active substances. A spray consisting of a 2% solution (w/v) of p-dimethylaminobenzaldehyde (PDAB) in 1.2 N hydrochloric acid and employed in the identification of allantoin and urea (1) also has proven useful in locating aureomycin and terramycin on paper chromatograms and in differentiating between these two antibiotics. After 5-8 hr. at room temperature, aureomycin shows ,up as a dirty yellow spot while terramycin gives a blue-green color. Approximately 5 pg. of the antibiotics applied over an area of 1 cm. in diameter is the smallest amount that can be detected. Chloromycetin, in quantities greater than 5 pg., can also be detected with this reagent, if the chromatogram is first sprayed with a 0.25% solution of stannous chloride in 1 N hydrochloric acid and allowed to dry. This procedure is a slight modification of the method of Smith and Worrel (2). After spraying with the PDAB reagent, a bright yellow spot appears after 5 min. at, room temperature. Qualitative reactions like these should increase the confidence with which the tentative identification of antibiotics on the basis of RI values in various solvent systems is regarded. REFERENCES 1. BERRY, H. K., SUTTON, H. E., CAIN, L., AND BERRY, J. S., Univ. Texas Publ. (Biochemical Institute Studies), No. IV (1951). 2. SMITH, G. N., AND WORREL, C. S., Arch. Biochem. 28, 1 (1959). W. T. SOKOLSKI HENRY KOFFLER P. A. TETRAULT
Laboratories Department
of Bacteriology, of Biological Sciences, Purdue University, Lafayette, Indiana Received December 1, 1969
Synthesis of Pantothenic Acid by Depyrophosphorylation Adenosine Triphosphate
of
Lipmann et al. (1) have recently reported that, in the enzymatic synthesis of acetyl-CoA, the energy transfer is effected by a pyrophosphate-liberating split of adenosine triphosphate (ATP). The synthesis of pantothenic acid, which previously was shown to proceed with ATP as energy source (2), now seems to involve a similar mechanism, evidenced by the appearance of inorganic pyrophosphate (PPi) and adenylic acid (AMP) as end products. This finding indicates a more general occurrence of depyrophosphorylation in energy transfer, especially since the synt.hesis of pantothenic acid does not seem to involve coenzyme A (CoA) (2). An experiment demonstrating the balance of reactants and products is shown in Table I. The enzyme used for this experiment was extrscted.from acetone1 Supported
in part by a grant from the Squibb Institute
for Medical Research.
LETTERS
TO
THE
237
EDITORS
dried cells of E’scherichia coli as described previously (2) and freed from most of the contaminating adenosinetriphosphatase (ATPase), myokinase, and pyrophosphatase by protamine and ammonium sulfate precipitations. Pantothenate was determined by microbiological assay with a mutant of E. coli (3); AMP spectrophotometrically according to Kalckar (4) using Schmidt’s deaminase; pyrophosphate as easily hydrolyzable phosphate after removal of ATP with charcoal (Norit) (5) ; ATP by difference in easily hydrolyzable phosphate before and after charcoal adsorption; inorganic orthophosphate (Pi) by the FiskeSubbaRow method (6). The identity and amount of pyrophosphate was checked further by treatment with five times recrystallized pyrophosphatase (7), kindly supplied by Dr. Kunitz, by acid hydrolysis of the Mn salt (8), and by paper chromatography (9); pantothenate, AMP, and ATP were also identified by paper chromatography in several solvent systems. TABLE
I
Balance of Reactants and Products Each tube received: 40 rmoles &alanine, 40 pmoles potassium pantoate, 40 pmoles ATP, 200 pmoles KCl, 40 pmoles MgClr, 200 pmoles KHCOI (pH 8.1), in a total volume of 2.0 ml. To each vessel, 0.1 ml. of extract containing 0.72 mg. protein was added. Incubation was at 37” for 80 min. At the beginning and the end of incubation samples were removed, inactivated by heating or treatment with trichloroacetic acid, and analyzed. Reaction mixture
Initial Final
Pantothenate pmoler/ml.
0.05 9.4
AMP pmolcs/ml.
@tOlES/9d.
PPi
0.3 9.5
0 7.2
Pi
ATP
@&.r/?nl.
pWlCS/d.
1.2 2.2
19.9 11.2
These data show a stoichiometric relationship between the amount of pantothenate synthesized and that of ATP split into AMP and pyrophosphate, suggesting the following over-all reaction : &alanine
+ pantoic
acid + ATP -+ pantothenic
acid + AMP + PPi
In support of the specific nature of the splitting of ATP, it was found that adenosine diphosphate (ADP) does not substitute for ATP as energy source. REFERENCES
F., JONES, M. E., BLACK, S., AND FLYNN, R. M., J. Am. Chem. 74, 2384 (1952). MAAS, W. K., J. Biol. Chem. 198,23 (1952). MAAS, W. K., AND DAVIS, B. D., J. Bact. 60, 733 (1950). KALCKAR, H. M., J. Biol. Chem. 167,445 (1947). CRANE, R. K., AND LIPMANN, F., J. Biol. Chem., in press. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 68,375 (1925).
1. LIPMANN,
2. 3. 4. 5. 6.
SOC.
238
LETTERS
7.
KUNITZ,
8. 9.
K?RNB.ERG, A., J. Biol. EBEL, J., AND VOLMAR,
THE
EDITORS
M., J. Gen. Physiol. 36, 423 (1962). Chem. 162, 779 (1950). Y., Compt. rend. 233,416
Biochemical Research Massachusetts General logical
TO
(1951).
Laboratory, Hospital
WERNER
and the Department
of Bio-
K.
G. DAVID
MAAS’ NOVELLI
Chemistry,
Harvard Medical School, Boston, Received January 21, 1965
Massachusetts
Paper Chromatography
of Some Ketoheptosesl
Klevstrand and Nordal (1) indicated that a spray reagent containing orcinol and trichloroacetic acid in water-saturated butanol was apparently specific for identifying ketoheptoses (sedoheptulose and mannoheptulose) on paper chromatograms. Bevenue and Williams (2) confirmed this observation with D-glucoTABLE Paper Chromatographic Sugar
n-Glucoheptulose n-Mannoheptulose n-Galaheptulose Sedoheptulose n-Idoheptulose L-Guloheptulose Sedoheptulosan . n-Idoheptulosan L-Guloheptulosan
Behavior RI (phenol)
0.42 0.40 0.48 0.46 0.60 0.53 0.70 0.64 0.65
I of Some
Ketoheptoses
Distance from origin (ethyl acetate) nm.
103 108 99 125 147 138 153 194 188
Color
with spray
reagent
Blue Green-blue Blue Blue Blue Blue Blue Blue Blue
heptulose in addition to sedoheptulose and mannoheptulose but further stated that sedoheptulosan did not give a color with the spray reagent. This latter observation was not in agreement with results on sedoheptulosan samples run in this laboratory, so some additional work was attempted. Through the courtesy of Dr. Nelson K. Richtmyer of the National Institutes of Health, Bethesda, Maryland, a number of ketoheptose samples were obtained, which included n-glucoheptulose, n-mannoheptulose, L-galaheptulose, sedoheptulosan monohydrate, n-idoheptulosan, and L-guloheptulosan hemihydrate. The last three samples were anhydrides, and to obtain a mixture of the free sugar and anhydride the anhydrides were treated with hot 0.2 N HCl as was described for the preparation of n-idoheptulose and n-idoheptulosan (3). The free sugars and anhydrides 1 Senior Asst. Scientist, U. S. Public Health Service. 1 Work performed under Contract No. W-7406-eng-26 Energy Commission.
for the U. S. Atomic