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[53] Aspartase
386
ENZYMES OF PROTEIN METABOLISM
[53]
[53] Aspartase L-Aspartic Acid ~ Fumaric Acid -~ NH3
(1)
By ARTTURI I. VIRTANEN and NILS ELLFOLK Assay Method
Principle. The method is based on the measuring of the amount of ammonia liberated. Reagents Stock solution of L-aspartic acid (0.1 M), pH 7.2. Phosphate buffer (M/15), pH 7.2. Enzyme preparation with initial velocity of 2 to 5 % NH3-N per minute per 10 ml.
Procedure. Two milliliters of aspartic acid solution, 2 ml. of phosphate buffer, and 6 ml. of distilled (0,3 ml. of toluene as antiseptic) water are incubated at 37 ° . Two-milliliter samples are taken for ammonia determination in an apparatus of Pucher et al. The ammonia is trapped in the receiver by 0.01 N H2S04 and is titrated iodometrically. The solution to be analyzed is made alkaline in the distillation flask with sodium carbonate buffer (5 g. of Na2C03 + 5 g. of NaC1 in 100 ml. of distilled water). Initial Velocity. Initial velocity is graphically obtained from the completed experimental curve and expressed in micrograms of NH3-N formed at 37 ° per minute at the pH and in the buffer designated. Purification Procedure 1
A. Propionic Acid Bacteria The nutrient solution for the propionic acid bacteria is prepared as follows. Fifty liters of skim milk is coagulated at 35 ° with rennet, heated to 96 ° to destroy the enzyme, filtered, and sterilized at 120 ° . After chilling to 45 ° the whey is inoculated with Lactobacillus helveticus. The lactic acid produced is neutralized with sterilized chalk. The whey culture is incubated at 42 ° until all the lactose is fermented to lactic acid, which is Checked with the Fehling test. The incubation has to be continued for 5 to 6 days. To 50 1. of fermented whey 1000 g. of pressed yeast and 100 g. of peptone are added. The mixture is heated to 120° for 30 minutes in an autoclave and centrifuged. The pH is adjusted to 6.8, and the solution is portioned out into 8-1. Erlenmeyer flasks and sterilized at 1 N. Ellfolk, Acta Chem. Scand. 7, 824 (1953).
[~3]
ASPARTASE
387
120 ° for 30 minutes. Before the inoculation of the propionic acid bacteria, the p H of the solution is controlled and adjusted to 6.8. The culture of the bacterial mass is performed in these Erlenmeyer flasks for the first 20 hours at 37 ° and then for 25 hours at 25 ° . The bacteria are separated from the solution with an air-driven Sharples supercentrifuge. The washed mass is dried on porous plates and ground to a dustlike powder. Preparation of Crude Extract. The dry bacteria are stirred thoroughly with n-butanol at a low temperature (0 to - 2 °) for 30 minutes and then separated by centrifuging at a low temperature ( - 2 °) and suspended in water at 0 °. The butanol is removed by dialyzing against tap water of low temperature ( + 2 ° ) . After 2~ hours of dialysis the bacteria are centrifuged off, the aspartase is obtained in a cell-free solution. Nucleic Acid Precipitation. By lowering the p H of the crude enzyme solution with 5 % acetic acid to 4.5-4.3, a strong opalescence was formed. After centrifugation the precipitate was easily dissolved when neutralized with solid CaCO3 (pH 6.5). Excess carbonate was removed by centrifugation. A quantitative recovery of aspartase was obtained in a colorless solution which has a high concentration of nucleic acids. 1~
B. Pscudomonas In culturing Ps. fluorescens a nutrient solution of the following composition is employed, at pH 7:100 1. of tap water, 800 g. of meat extractpeptone powder (Baeto Nutrient Broth Dehydrated, Difco Laboratories), 300 g. of K2HPOt, 100 g. of MgS04-7 H20. The culture of the bacterial mass is performed in large vats or in Roux flasks at 25 °. After 48 hours the bacteria are harvested b y means of a milk separator and washed with tap water. The bacterial mass is dried on porous plates, and the dry mass is ground to a dustlike powder. Preparation of Crude Extract. The extract is prepared with n-butanol in exactly the same way as described above. Aspartase is precipitated from the extract b y lowering the p H to 4.5 with 0.1 M acetic acid at 0 °. The precipitate should immediately be dissolved in neutral buffer (m/15 phosphate, pH 7.2).
Properties Specificity. The enzyme is strictly specific to L-aspartie acid at~d fumarie acid, having no action on D-aspartic acid, 2 L-cysteic acid, 3 a,$-diaminosuccinic acid, 3 or other amino acids tested (glycine, 4 alanine, ~ leux, N. Ellfolk, Acta Chem. Scand. 9, in press (1955). 2 A. I. V i r t a n e n a n d T. Laine, Suomen Kemistilehti B9, 28 (1936). 3 N. Ellfolk, Acta Chem. Scand. 8, 151 (1954). 4 j . H. Quastel a n d B. Woolf, Biochem. J. 20, 545 (1926). 5 R. P. Cook a n d B. Woolf, Biochem. J. 22, 474 (1926).
388
ENZYMES OF PROTEIN METABOLISM
[53]
cine, ~ phenylalanine, 7 tyrosine, 7 dioxyphenylalanine, 7 histidine, 7 glutamic acid4). Ammonia was not added to maleic, 4 glutaconic, 4 crotonic, 8 mesaconic, ° aconitic, ~ sorbic acid, ~ or the diamide 6 and mono -9 and diethyP ester of fumaric acid. Inhibitors. 1° T h e enzyme of propionic acid bacteria is inhibited by citrate, oxalate, and ethylenediaminetetraacetic acid and pyrophosphate. A weak inhibition is observed with cyanide, azide, and acetonitrile. Practically no inhibition is produced b y fluoride and sodium sulfide. A metal seems to be responsible for the activity of this enzyme. As to the thiol group reagents, strong inhibition power is observed with p-chloromercuribenzoate, whereas weaker effects are observed with oxidizing agents (ferricyanide, o-iodosobenzoate, iodine) and different arsenicals (phenylarsine oxide and 3-amino-4-hydroxydichloroarsine hydrochloride). Iodoacetamide produced a rather weak inhibition. BAL (2,3-dimercaptopropanol) has an antidotal effect on the action of p-cloromercuribenzoate. Certain h e a v y metal ions inhibit the enzyme. Strong inhibitory effect is observed with Ag, Hg, Zn, Cd, and Co. A weaker inhibition is observed with Pb and Ni. These observations point to the existence of an essential thiol group in the enzyme. Effect of pH. The enzyme exhibits a sharp optimum for activity at p H 7.5 in phosphate buffer. 8
Distribution Aspartase is a typical bacterial enzyme, being present, e.g., in Escherichia coli,4 Pseudomonas fluorescens,5 Ps. pyocyaneus,5 Ps. aeruginosa,1 Proteus vulgaris, 5 Serratia marcescens, 5 Propionibacterium sp., 6 Lactobacillus helveticus, ~ Diplococcus pneumoniae, ix Micrococcus aureus, ll Sarcina spp., 11 Salmonella enteriditis. ~1 B o t t o m yeast, 12 brewer's yeast, 1~ and molds (PeniciUium notatum) ~ also have aspartase activity. In higher plants 8 and their seedlings 1~ the activities are weak or cannot be demonstrated. No definitive evidence of the presence of this enzyme in animal tissues has been reported. 5 s A. I. V i r t a n e n a n d J. T a r n a n e n , Bioehem. Z. 250, 193 (1932). K. P. Jacobsohn a n d M. Soares, Compt. rend. soc. biol. 125, 554 (1937). s T. Ichihara, Hukuoka Acta Med. 24, 1231 (1931) [Chem. Abstr. 9.6, 3539 (1932)].
9 K. P. Jacobsohn and M. Soares, Enzymologia 1, 183 (1936). 10N. Ellfolk, Acta Chem. Scand. 7, 1155 (1953). 11H. Saito, J. Biochem. (Japan) 34, 49, 103 (1941) [Chem. Abstr. 4[i, 1199 (1951)]. II H. Haehn and H. Leopold, Biochem. Z. 292, 380 (1937). is y. Sumiki, Bull. Japan. Soc. Ferment. 23, 33 (1928) [Chem. Abstr. 23, 2531 (1929)]. 14N. Tsuda, Japan. J. Nutrition 8, 108 (1950) [Chem. Abstr. 45, 8591a (1951)]. 15 M. Damodaran and S. S. Subramanian, Proc. Indian Acad. Sci. 27B, 47 (1948).
[53]
ASPARTASE
389
Quantitative Determination of L-Aspartic Acid Aspartase can be used for the quantitative determination of L-aspartic acid, although the enzyme preparations contain fumarase and the ammonia formation depends not only on reaction 1 b u t also on reaction 2 Fumaric acid W H20 ~ Malic acid
(2)
Three parallel experiments are needed: (1) the actual experiment; (2) one to which has been added approximately the same a m o u n t of aspartic acid as is found in the solution to be investigated; and (3) an experiment with enzyme preparation alone. The following example illustrates the method (Virtanen and LouhivuorP6). Take six test tubes, containing 5 ml. of 0.067 M phosphate buffer (pH 7.0) and 2 ml. of toluene per tube. Suspend 500 rag. of dried powder of Ps. fluorescens carefully in each solution. Add to three of the tubes 10 ml. of solution with 20.0 mg. of neutralized L-aspartic acid, and to the other three 10 ml. of distilled water. I n c u b a t e at 37 ° with occasional shaking. After equilibrium is reached, place the tubes in ice water for interruption of the reaction.
Aspartic Expt. acid, mg. I II III
--
IV V VI
20 20 20
Aspartic acid-N, mg. ----
Average 2.10 2.10 2.10 Average
0.01 N NHs-N H2SO4 formed, ml. mg.
NH3-N NH3-N formed liberated Found from from aspartic aspartic aspartie acid-N, % acid, mg. acid-N, % of added ~
17.20 17.46 17.38
2.408 2.444 2.433
----
----
----
26.53 26.96 26.79
2.428 3.714 3.774 3.751
1.286 1.346 1.323
62.19 64.09 63.00
98.6 101.6 99.9
3.746
63.09
Calculated from the mean value of ammonia formed from aspartic acid. The accuracy of the ammonia determination decides how small an a m o u n t of L-aspartic acid can be determined b y the method. B y using dry preparations or strong enzyme solutions (amorphous powder after lyophilization is quite stable) the enzyme concentration can be raised so high t h a t the equilibrium is reached in some hours. 16A. I. Virtanen and A. Louhivuori, Acta Chem. Scand. 1, 799 (1947).
390
ENZYMES OF PROTEIN METABOLISM
[54]
Aspartase can well be applied to determination of L-aspartic acid in protein hydrolyzates. When determination is performed in solutions containing dicarboxylic acids (oxalic acid, citric acid, fumaric and malonic acids), ~° however, these acids must be removed from the solution before the determination, since, being inhibitors to aspartase, an equilibrium is established on the basis of which no calculations on the content of aspartic acid can be made.
[E4] A m i n e
Oxidases
A. Amine Oxidase from Steer Plasma ~ RCH2NH2 + 05--~ RCHO + H202 + NH3 B y CELIA WHITE TABOR, HERBERT TABOR, and
SANFORD M. ROSENTHAL Assay M e t h o d The oxidative deamination of amines is usually followed by the measurement of oxygen consumption or ammonia production. 2 The spectrophotometric assay used here involves the measurement of the benzaldehyde produced during the oxidative deamination of benzylamine and depends on the difference in the absorption spectrum of benzaldehyde and benzylamine at 250 m~. The molar extinction coefficient of benzaldehyde is 13,000, whereas that of benzylamine is <200. Reagents
0.2 M potassium phosphate buffer, pH 7.2. 0.1 M benzylamine sulfate. 1.07 g. of redistilled benzylamine and 5 ml. of 2 N I-I2S04 are made up to 100 ml. with distilled water. Enzyme. About 10 to 50 spectrophotometric units (see below) are used for each determination. Procedure. One milliliter of 0.2 M phosphate buffer, 0.1 ml. of 0.1 M benzylamine sulfate, enzyme, and water (final volume of 3.0 ml.) are placed in a silica cell having a 1-cm. light path. A blank cell is made up
1The method reported here has been described in J. Biol. Chem. 208, 645 (1954). 2Literature has been reviewed by E. A. Zeller in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. II, p. 536, Academic Press, New York; and by H. Blaschko, Pharmacol. Revs. 4, 415 (1952).
ENZYMES OF PROTEIN METABOLISM
[53]
[53] Aspartase L-Aspartic Acid ~ Fumaric Acid -~ NH3
(1)
By ARTTURI I. VIRTANEN and NILS ELLFOLK Assay Method
Principle. The method is based on the measuring of the amount of ammonia liberated. Reagents Stock solution of L-aspartic acid (0.1 M), pH 7.2. Phosphate buffer (M/15), pH 7.2. Enzyme preparation with initial velocity of 2 to 5 % NH3-N per minute per 10 ml.
Procedure. Two milliliters of aspartic acid solution, 2 ml. of phosphate buffer, and 6 ml. of distilled (0,3 ml. of toluene as antiseptic) water are incubated at 37 ° . Two-milliliter samples are taken for ammonia determination in an apparatus of Pucher et al. The ammonia is trapped in the receiver by 0.01 N H2S04 and is titrated iodometrically. The solution to be analyzed is made alkaline in the distillation flask with sodium carbonate buffer (5 g. of Na2C03 + 5 g. of NaC1 in 100 ml. of distilled water). Initial Velocity. Initial velocity is graphically obtained from the completed experimental curve and expressed in micrograms of NH3-N formed at 37 ° per minute at the pH and in the buffer designated. Purification Procedure 1
A. Propionic Acid Bacteria The nutrient solution for the propionic acid bacteria is prepared as follows. Fifty liters of skim milk is coagulated at 35 ° with rennet, heated to 96 ° to destroy the enzyme, filtered, and sterilized at 120 ° . After chilling to 45 ° the whey is inoculated with Lactobacillus helveticus. The lactic acid produced is neutralized with sterilized chalk. The whey culture is incubated at 42 ° until all the lactose is fermented to lactic acid, which is Checked with the Fehling test. The incubation has to be continued for 5 to 6 days. To 50 1. of fermented whey 1000 g. of pressed yeast and 100 g. of peptone are added. The mixture is heated to 120° for 30 minutes in an autoclave and centrifuged. The pH is adjusted to 6.8, and the solution is portioned out into 8-1. Erlenmeyer flasks and sterilized at 1 N. Ellfolk, Acta Chem. Scand. 7, 824 (1953).
[~3]
ASPARTASE
387
120 ° for 30 minutes. Before the inoculation of the propionic acid bacteria, the p H of the solution is controlled and adjusted to 6.8. The culture of the bacterial mass is performed in these Erlenmeyer flasks for the first 20 hours at 37 ° and then for 25 hours at 25 ° . The bacteria are separated from the solution with an air-driven Sharples supercentrifuge. The washed mass is dried on porous plates and ground to a dustlike powder. Preparation of Crude Extract. The dry bacteria are stirred thoroughly with n-butanol at a low temperature (0 to - 2 °) for 30 minutes and then separated by centrifuging at a low temperature ( - 2 °) and suspended in water at 0 °. The butanol is removed by dialyzing against tap water of low temperature ( + 2 ° ) . After 2~ hours of dialysis the bacteria are centrifuged off, the aspartase is obtained in a cell-free solution. Nucleic Acid Precipitation. By lowering the p H of the crude enzyme solution with 5 % acetic acid to 4.5-4.3, a strong opalescence was formed. After centrifugation the precipitate was easily dissolved when neutralized with solid CaCO3 (pH 6.5). Excess carbonate was removed by centrifugation. A quantitative recovery of aspartase was obtained in a colorless solution which has a high concentration of nucleic acids. 1~
B. Pscudomonas In culturing Ps. fluorescens a nutrient solution of the following composition is employed, at pH 7:100 1. of tap water, 800 g. of meat extractpeptone powder (Baeto Nutrient Broth Dehydrated, Difco Laboratories), 300 g. of K2HPOt, 100 g. of MgS04-7 H20. The culture of the bacterial mass is performed in large vats or in Roux flasks at 25 °. After 48 hours the bacteria are harvested b y means of a milk separator and washed with tap water. The bacterial mass is dried on porous plates, and the dry mass is ground to a dustlike powder. Preparation of Crude Extract. The extract is prepared with n-butanol in exactly the same way as described above. Aspartase is precipitated from the extract b y lowering the p H to 4.5 with 0.1 M acetic acid at 0 °. The precipitate should immediately be dissolved in neutral buffer (m/15 phosphate, pH 7.2).
Properties Specificity. The enzyme is strictly specific to L-aspartie acid at~d fumarie acid, having no action on D-aspartic acid, 2 L-cysteic acid, 3 a,$-diaminosuccinic acid, 3 or other amino acids tested (glycine, 4 alanine, ~ leux, N. Ellfolk, Acta Chem. Scand. 9, in press (1955). 2 A. I. V i r t a n e n a n d T. Laine, Suomen Kemistilehti B9, 28 (1936). 3 N. Ellfolk, Acta Chem. Scand. 8, 151 (1954). 4 j . H. Quastel a n d B. Woolf, Biochem. J. 20, 545 (1926). 5 R. P. Cook a n d B. Woolf, Biochem. J. 22, 474 (1926).
388
ENZYMES OF PROTEIN METABOLISM
[53]
cine, ~ phenylalanine, 7 tyrosine, 7 dioxyphenylalanine, 7 histidine, 7 glutamic acid4). Ammonia was not added to maleic, 4 glutaconic, 4 crotonic, 8 mesaconic, ° aconitic, ~ sorbic acid, ~ or the diamide 6 and mono -9 and diethyP ester of fumaric acid. Inhibitors. 1° T h e enzyme of propionic acid bacteria is inhibited by citrate, oxalate, and ethylenediaminetetraacetic acid and pyrophosphate. A weak inhibition is observed with cyanide, azide, and acetonitrile. Practically no inhibition is produced b y fluoride and sodium sulfide. A metal seems to be responsible for the activity of this enzyme. As to the thiol group reagents, strong inhibition power is observed with p-chloromercuribenzoate, whereas weaker effects are observed with oxidizing agents (ferricyanide, o-iodosobenzoate, iodine) and different arsenicals (phenylarsine oxide and 3-amino-4-hydroxydichloroarsine hydrochloride). Iodoacetamide produced a rather weak inhibition. BAL (2,3-dimercaptopropanol) has an antidotal effect on the action of p-cloromercuribenzoate. Certain h e a v y metal ions inhibit the enzyme. Strong inhibitory effect is observed with Ag, Hg, Zn, Cd, and Co. A weaker inhibition is observed with Pb and Ni. These observations point to the existence of an essential thiol group in the enzyme. Effect of pH. The enzyme exhibits a sharp optimum for activity at p H 7.5 in phosphate buffer. 8
Distribution Aspartase is a typical bacterial enzyme, being present, e.g., in Escherichia coli,4 Pseudomonas fluorescens,5 Ps. pyocyaneus,5 Ps. aeruginosa,1 Proteus vulgaris, 5 Serratia marcescens, 5 Propionibacterium sp., 6 Lactobacillus helveticus, ~ Diplococcus pneumoniae, ix Micrococcus aureus, ll Sarcina spp., 11 Salmonella enteriditis. ~1 B o t t o m yeast, 12 brewer's yeast, 1~ and molds (PeniciUium notatum) ~ also have aspartase activity. In higher plants 8 and their seedlings 1~ the activities are weak or cannot be demonstrated. No definitive evidence of the presence of this enzyme in animal tissues has been reported. 5 s A. I. V i r t a n e n a n d J. T a r n a n e n , Bioehem. Z. 250, 193 (1932). K. P. Jacobsohn a n d M. Soares, Compt. rend. soc. biol. 125, 554 (1937). s T. Ichihara, Hukuoka Acta Med. 24, 1231 (1931) [Chem. Abstr. 9.6, 3539 (1932)].
9 K. P. Jacobsohn and M. Soares, Enzymologia 1, 183 (1936). 10N. Ellfolk, Acta Chem. Scand. 7, 1155 (1953). 11H. Saito, J. Biochem. (Japan) 34, 49, 103 (1941) [Chem. Abstr. 4[i, 1199 (1951)]. II H. Haehn and H. Leopold, Biochem. Z. 292, 380 (1937). is y. Sumiki, Bull. Japan. Soc. Ferment. 23, 33 (1928) [Chem. Abstr. 23, 2531 (1929)]. 14N. Tsuda, Japan. J. Nutrition 8, 108 (1950) [Chem. Abstr. 45, 8591a (1951)]. 15 M. Damodaran and S. S. Subramanian, Proc. Indian Acad. Sci. 27B, 47 (1948).
[53]
ASPARTASE
389
Quantitative Determination of L-Aspartic Acid Aspartase can be used for the quantitative determination of L-aspartic acid, although the enzyme preparations contain fumarase and the ammonia formation depends not only on reaction 1 b u t also on reaction 2 Fumaric acid W H20 ~ Malic acid
(2)
Three parallel experiments are needed: (1) the actual experiment; (2) one to which has been added approximately the same a m o u n t of aspartic acid as is found in the solution to be investigated; and (3) an experiment with enzyme preparation alone. The following example illustrates the method (Virtanen and LouhivuorP6). Take six test tubes, containing 5 ml. of 0.067 M phosphate buffer (pH 7.0) and 2 ml. of toluene per tube. Suspend 500 rag. of dried powder of Ps. fluorescens carefully in each solution. Add to three of the tubes 10 ml. of solution with 20.0 mg. of neutralized L-aspartic acid, and to the other three 10 ml. of distilled water. I n c u b a t e at 37 ° with occasional shaking. After equilibrium is reached, place the tubes in ice water for interruption of the reaction.
Aspartic Expt. acid, mg. I II III
--
IV V VI
20 20 20
Aspartic acid-N, mg. ----
Average 2.10 2.10 2.10 Average
0.01 N NHs-N H2SO4 formed, ml. mg.
NH3-N NH3-N formed liberated Found from from aspartic aspartic aspartie acid-N, % acid, mg. acid-N, % of added ~
17.20 17.46 17.38
2.408 2.444 2.433
----
----
----
26.53 26.96 26.79
2.428 3.714 3.774 3.751
1.286 1.346 1.323
62.19 64.09 63.00
98.6 101.6 99.9
3.746
63.09
Calculated from the mean value of ammonia formed from aspartic acid. The accuracy of the ammonia determination decides how small an a m o u n t of L-aspartic acid can be determined b y the method. B y using dry preparations or strong enzyme solutions (amorphous powder after lyophilization is quite stable) the enzyme concentration can be raised so high t h a t the equilibrium is reached in some hours. 16A. I. Virtanen and A. Louhivuori, Acta Chem. Scand. 1, 799 (1947).
390
ENZYMES OF PROTEIN METABOLISM
[54]
Aspartase can well be applied to determination of L-aspartic acid in protein hydrolyzates. When determination is performed in solutions containing dicarboxylic acids (oxalic acid, citric acid, fumaric and malonic acids), ~° however, these acids must be removed from the solution before the determination, since, being inhibitors to aspartase, an equilibrium is established on the basis of which no calculations on the content of aspartic acid can be made.
[E4] A m i n e
Oxidases
A. Amine Oxidase from Steer Plasma ~ RCH2NH2 + 05--~ RCHO + H202 + NH3 B y CELIA WHITE TABOR, HERBERT TABOR, and
SANFORD M. ROSENTHAL Assay M e t h o d The oxidative deamination of amines is usually followed by the measurement of oxygen consumption or ammonia production. 2 The spectrophotometric assay used here involves the measurement of the benzaldehyde produced during the oxidative deamination of benzylamine and depends on the difference in the absorption spectrum of benzaldehyde and benzylamine at 250 m~. The molar extinction coefficient of benzaldehyde is 13,000, whereas that of benzylamine is <200. Reagents
0.2 M potassium phosphate buffer, pH 7.2. 0.1 M benzylamine sulfate. 1.07 g. of redistilled benzylamine and 5 ml. of 2 N I-I2S04 are made up to 100 ml. with distilled water. Enzyme. About 10 to 50 spectrophotometric units (see below) are used for each determination. Procedure. One milliliter of 0.2 M phosphate buffer, 0.1 ml. of 0.1 M benzylamine sulfate, enzyme, and water (final volume of 3.0 ml.) are placed in a silica cell having a 1-cm. light path. A blank cell is made up
1The method reported here has been described in J. Biol. Chem. 208, 645 (1954). 2Literature has been reviewed by E. A. Zeller in "The Enzymes" (J. B. Sumner and K. Myrb~ck, eds.), Vol. II, p. 536, Academic Press, New York; and by H. Blaschko, Pharmacol. Revs. 4, 415 (1952).