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Proline synthesis in Escherichia coli a proline-inhibitable glutamic acid kinase
462 BBA
BIOCHIMICA
ET BIOPHYSICA
ACTA
26 245
PROLINE
SYNTHESIS
IN ESCHERICHIA
X PROLINE-INHIBITABLE ANNETTE
COLI
GLUTAMIC
ACID
KINASE
BAICH*
Department of Biochemistry and Biophysics, Oregon State University, Cowallis, Owg. 97331 (Received
(C..S..-l.)
July 4th, 1969)
SUMMARY
The object
of this work was the identification
of the first enzyme
in the bio-
synthesis of proline in Escherichia coli. A glutamic acid-dependent reaction, which releases Pi from ATP and which is inhibitable by proline, was identified in extracts of these bacteria. The reaction in vitro is dependent upon the presence of imidazole. The reaction was implicated in proline biosynthesis on the basis of its sensitivity to inhibition by proline and because extracts of cells not subject to end-product hibition in vivo catalyzed a reaction which was not inhibited i?z vitro.
in-
INTRODUCTION
It
has
been
suggested1-3
that
proline
is synthesized
Escherichia coli by the following series of reactions
from glutamic
acid in
:
Glutamic acid
+ L4TP x+
ADP + PI + (glutamyl-enzyme)
(Glutamyl-enzyme)
+ reducing agent + glutamic y-semialdehyde
(2)
Glutamic y-semialdehyde
+ NADPH
(3)
+ NADP+
+ proline
(I)
The action of the first enzyme in the pathway would be similar to that found and possibly that of glutathione5. A problem thus in the synthesis of glutamine arises in distinguishing the first reaction in the biosynthesis of proline from other similar enzyme activities. This problem is solved by taking advantage of the extreme sensitivity of the proline pathway to end-product inhibitior9. Consequently, the first enzyme in the pathway should not only catalyze a glutamic acid-dependent hydrolysis of ATP, but this reaction should be inhibited by proline. A reaction of this kind has been identified in extracts of E. cob. In addition to the predicted properties of the enzyme, a requirement for imidazole was found. MATERIALS
AND METHODS
Deoxyribonuclease was obtained from the Worthington Biochemical Corp. and had an activity of 2650 units/mg. Glutamic acid was obtained from the Mann * Present address: Southern Illinois University, Edwardsville, Biochim. Biophys. Acta, 192 (1969) 462-467
Ill. 62025, 1J.S.h.
PROLINE
SYNTHESIS
IN
E.
463
coli
Co. ATP and Grade I crystalline
imidazole were obtained
from Sigma. Bovine
serum
albumin was a product of the Pentex Corporation. Bacterial strains. Strain 55-x is derived from Escherichia coli W and requires proline for growth because it lacks pyrroline-5-carboxylate reductase (Enzyme 3 in the pathway). Strain WPI does not require proline for growth, but lacks control of its proline biosynthetic pathways. Preparation of the enzyme. Bacteria were grown to the end of log phase in a 100-1 stainless-steel
fermentor
in an inorganic
salts
medium,
supplemented
with
IOO pg/ml proline when required. The cells were harvested by centrifugation, washed twice with distilled water and frozen at -20~. Frozen paste was dispersed in distilled water and passed through an Eaton press’. The frozen extracts were stored at -20~. Frozen extract (13 g) was dispersed in z5 ml of distilled water until thawed. Deoxyribonuclease (0.5 ml, I mg/ml) was added and the mixture stirred gently until the viscosity of the solution diminished to that of water. All subsequent procedures were carried out at 4’. The extract was centrifuged once at 13 ooo rev./min for IO min to remove the unbroken cells and cell debris and a second time for 60 min at the same speed to remove other particulate matter. The crude extract was treated with onetenth its volume of 20% streptomycin sulfate to precipitate nucleic acids, centrifuged for IO min to remove the nucleic acid precipitate, and the supernatant solution was dialyzed overnight against distilled water. During this dialysis, a heavy precipitate formed inside the dialysis bag. This precipitate, which contained the enzymatic activity, was recovered by centrifugation, taken up in 20 ml of distilled water and homogenized. Unless otherwide specified, this homogenate was used as the enzyme in these studies. Analytical procedures. Activity of the enzyme was estimated from the release of Pi from ATP. The assay mixture
contained,
in a volume of 0.2 ml, ~-IO-~ M ATP,
0.1 M glutamic acid (pH 7.6), 5.10-~ M MgCl, and 2.5.10-~ M imidazole buffer (pH 7.6) and enzyme. One unit of activity was defined as that amount of enzyme which caused the proline-inhibitable
release of I pmole of Pi per h. Pi was measured
using the reagent of FISKE and SUBBAROW~ after the samples had been deproteinized with 2.5% trichloroacetic acid. Activation of glutamate was estimated by the hydroxamate method of LIP~IANN AND TUTTLE~. Protein was measured by the method of LOWRY et aLlo, using crystalline bovine serum albumin as the standard. RESULTS
A 7.7-fold purification of the enzymatic activity with little loss of activity was achieved (Table I). The release of Pi from ATP requires glutamic acid, MgCl, and imidazole and is inhibited by proline (Table II). In the best preparation, the precipitate obtained upon dialysis after streptomycin treatment, 80 y0 of the release of Pi by the extract was inhibitable by I - IO-~ M proline. Marked substrate activation is observed when the concentration of glutamate exceeds 0.05 M. Below this concentration, a straight line Michealis-Menten curve is obtained with a Km for glutamate of 0.007-0.01 M. The addition of I-IO-~ M proline to the assay mixture increases the apparent Km for glutamic acid, as well as producing B&him.
Biophys.
Acta,
192 (1969) 462-467
A.
464
BAICH
a nonlinear response (Fig. I). Higher concentrations of proline inhibit the reaction almost completely (Fig. 2). Maximum activity of the enzyme was found between pH 7.6 and 8.6 (Fig. 3). At higher pH values. inhibition of the reaction by proline was somewhat overcome. The requirement for imidazole is illustrated in Fig. 4, and low concentrations TABLE
I
PURIFICATION
OF GLUTAMIC
Y-SEMIALDEHYDE
Activity (units)
Crude extract After streptomycin treatment Precipitate after dialysis
TABLE
‘37 151
109
SYNTHETASE
Protein
.SpCCi& activity
(md 2 790
0.0~8
I550
0.097
297
Purification
Recovery (%) IO0
2.0
0.37
7.7
II0
81
II
REQUIREMENT
FOR THE
ENZYMATIC
RELEASE
OF Pi BY
THE
ENZYRlE
The complete system contains, in o.z-ml final volume, 7’ IO-~ M ATP, 0.1 M glutamic acid (pH 7.6), 5’ 10-2 M M&l,, 2,5’ 10-2 M imidazole (pH 7.6) and enzyme. Systenz
Release Strain
Complete + proline (1. ro-3 M) -glutamate -imidazole
IO0 5.9 0 II.8
(% ) Strain
83.7 3.8 16.6
0
0
-M&l,
3.5
0
0
0
I
I
50
OO I.
Biochim.
I
l/S] CM-‘)
100
WPr
100
-enzyme
- ATP
Fig.
of P< 5 j-I
I
150
I
Effect of substrate concentration on velocity of reaction. Samples assayed in the absence resence (A) of I. IO-~ M proline. Concentration of protein was 0.12 mg/o.z ml reaction
Biophys.
Acta,
192
(1969) 462-467
PROLINE
SYNTHESIS
IN
E.
465
COh
Fig. 2. Effect of proline on the velocity volume of reaction mixture.
of reaction.
Concentration
of protein was 0.16 mg per 0.2 ml
Fig. 3, Effect of pH on the velocity of reaction. Samples assayed in the absence (0) and presence (a ) of I. 10-5M proline. Concentration of protein was 0.16 mg/o.a ml volume of reaction mixture.
0 Imidozole
0.0 5
concn.
0.1
CM)
Fig. 4. Effect of imidazole on the velocity of reaction. Samples tested in the absence (0) and presence (a ) of I. IO-~ M proline. Concentration of protein 0.12 mg/o.z ml reaction mixture. TABLE
III
A COMPARISON
OF HYDROXAMIC
ACID
system
Pt released (pmoles)
2 M NH,OH + I. IO-~M proline
7.5 2.5
IO-~
+ I.
M imidazole M proline
IO-~
FORMED
AND
THE
P,
RELEASED
Hydroxamate formed (,umoZes) 6.0
-
5.0 5.
DERIVATIVE
1.0
-
5.0
6.6 I.3 5.3
Biochim. Biophys. Acta, 192 (1969) 462.-467
A. BAICH
466
of proline increase the dependence of the reaction on this compound. Activation of glutamic acid was demonstrated by the formation of a hydroxamic acid derivative. The formation of this derivative was not dependent upon imidazole; the presence of hydroxylamine permits the release of Pr (Table III). DIscussIon:
The rate of the reaction in extracts prepared from a controlled strain of bacteria is a sensitive function of the concentrations of glutamic acid, proline and imidazole. Extracts of strain WPI, which is not subject to end-product inhibition of proline biosynthesis in vivo, catalyzes a corresponding reaction which is less sensitive to the action of proline. The substrate dependence on the rate of reaction shows a cooperative effect only at very high glutamate concentrations under the conditions used here. However, the addition of a low concentration of proline gives the nonlinear Michaelis-Menten curve characteristic of an allosteric effector. Because of the lability of free y-glutamyl phosphaten, an enzyme-bound intermediate is probably involved in the reaction. Two structures may be proposed for this intermediate, y-glutamyl phosphate, which is analogous to products formed in the aspartokinase12 and N-acetylglutamokinase13 reactions, and y-glutamyl enzyme, which is similar to the complex observed in the synthesis of glutamine4. The reaction may occur by way of one or both of these intermediates (Fig. 5). Imidazole and hydroxylamine are considered to cause a nucleophilic displacement of the enzyme from the enzyme-substrate complex14y15. It is not proposed that the imidazole-dependent mechanism operates in vivo but that added imidazole acts as a model or substitute for a subsequent enzyme reaction. The second reaction of the biosynthetic pathway, which catalyzes the formation of glutamic y-semialdehyde, would serve this purpose in vivo. Because the substrate is apparently enzyme (55-1)
Cd0 , ‘OH YNH2
+ATP + Enzyme
tdg2+
_
40
C, I OH
HC-NH2 Enzyme
+
CH2
CH2
CH2 $0, *NOH
Fig. 5. Proposed mechanism for the activation of glutamic acid. Biochim.
Biophys.
Acta,
192 (1969)
462-467
f
Enzyme
PROLINE
SYNTHESIS
IN
E.
467
COli
bound, it could be anticipated that the second enzyme in the pathway is close to, or bound to, the first in whole cells. The reaction described here is implicated in the biosynthesis of proline on the basis of its sensitivity to inhibition by proline, in contrast to glutamine synthetase16, and because extracts of cells which excrete proline in vivo contain a corresponding enzyme which is not inhibited by this compound. ACKNOWLEDGMENTS
This investigation was supported by Public Health Service Grant No. CA-ozzgg, from the National Cancer Institute. I am the recipient of a Research Career Development award from the Public Health Service, National Institute of Allergy and Infectious Diseases. The technical assistance of Mrs. Linda Haley is gratefully acknowledged. REFERENCES I 2 3 4
STRECKER, J. Biol. Chem., 225 (1957) 825. VOGEL, P. H. ABELSON AND E. T. BOLTON, Biochim. Biophys. Acta., II (1953) 584. VOGEL AND B. D. DAVIS, J. Am. Chem. Sot., 74 (1952) Iog. KRISHNASWAMY, V. PAMILJANS AND A. MEISTER, J. Biol. Chem., 237 (1962) 2932. JOHNSTON AND K. BLOCH, J. Biol. Chem., 188 (1951) 221. A. BAICH AND D. J. PIERSON, Biochim. Biophys. Acta., 104 (1965) 397. N. R. EATON, J. Bacteriol., 83 (1962) 1359. C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375. F. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (1945) 21. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. A. KATCHALSKY AND M. PAECHT, J. Am. Chem. Sot., 76 (1954) 6042. S. BLACK AND N. G. WRIGHT, J. Biol. Chem., 213 (1955) 27. A. BAICH AND H. J. VOGEL, Biochem. Biophys. Res. Commun., 7 (1962) 491. M. L. BENDER AND B. W. TURNQUEST, J. Am. Chem. SOL, 79 (1957) 1652. M. L. BENDER AND B. W. TURNQUEST, J. Am. Chem. Sot., 7g (1957) 1656. T. C. BRUICE AND G. L. SCHMIR, J. Am. Chem. SOL, 79 (1957) 1663. C. A. WOOLFOLK AND E. R. STADTMAN, Biochem. Biophys. Res. Commun., 17 (1964) 313.
H. H. H. P. 5 R. 6 7
8 9 IO II
12 13 14 15 16
17
J. J. J. R. B.
Biochim.
Biophys.
Acta,
192 (1969) 462-467
BIOCHIMICA
ET BIOPHYSICA
ACTA
26 245
PROLINE
SYNTHESIS
IN ESCHERICHIA
X PROLINE-INHIBITABLE ANNETTE
COLI
GLUTAMIC
ACID
KINASE
BAICH*
Department of Biochemistry and Biophysics, Oregon State University, Cowallis, Owg. 97331 (Received
(C..S..-l.)
July 4th, 1969)
SUMMARY
The object
of this work was the identification
of the first enzyme
in the bio-
synthesis of proline in Escherichia coli. A glutamic acid-dependent reaction, which releases Pi from ATP and which is inhibitable by proline, was identified in extracts of these bacteria. The reaction in vitro is dependent upon the presence of imidazole. The reaction was implicated in proline biosynthesis on the basis of its sensitivity to inhibition by proline and because extracts of cells not subject to end-product hibition in vivo catalyzed a reaction which was not inhibited i?z vitro.
in-
INTRODUCTION
It
has
been
suggested1-3
that
proline
is synthesized
Escherichia coli by the following series of reactions
from glutamic
acid in
:
Glutamic acid
+ L4TP x+
ADP + PI + (glutamyl-enzyme)
(Glutamyl-enzyme)
+ reducing agent + glutamic y-semialdehyde
(2)
Glutamic y-semialdehyde
+ NADPH
(3)
+ NADP+
+ proline
(I)
The action of the first enzyme in the pathway would be similar to that found and possibly that of glutathione5. A problem thus in the synthesis of glutamine arises in distinguishing the first reaction in the biosynthesis of proline from other similar enzyme activities. This problem is solved by taking advantage of the extreme sensitivity of the proline pathway to end-product inhibitior9. Consequently, the first enzyme in the pathway should not only catalyze a glutamic acid-dependent hydrolysis of ATP, but this reaction should be inhibited by proline. A reaction of this kind has been identified in extracts of E. cob. In addition to the predicted properties of the enzyme, a requirement for imidazole was found. MATERIALS
AND METHODS
Deoxyribonuclease was obtained from the Worthington Biochemical Corp. and had an activity of 2650 units/mg. Glutamic acid was obtained from the Mann * Present address: Southern Illinois University, Edwardsville, Biochim. Biophys. Acta, 192 (1969) 462-467
Ill. 62025, 1J.S.h.
PROLINE
SYNTHESIS
IN
E.
463
coli
Co. ATP and Grade I crystalline
imidazole were obtained
from Sigma. Bovine
serum
albumin was a product of the Pentex Corporation. Bacterial strains. Strain 55-x is derived from Escherichia coli W and requires proline for growth because it lacks pyrroline-5-carboxylate reductase (Enzyme 3 in the pathway). Strain WPI does not require proline for growth, but lacks control of its proline biosynthetic pathways. Preparation of the enzyme. Bacteria were grown to the end of log phase in a 100-1 stainless-steel
fermentor
in an inorganic
salts
medium,
supplemented
with
IOO pg/ml proline when required. The cells were harvested by centrifugation, washed twice with distilled water and frozen at -20~. Frozen paste was dispersed in distilled water and passed through an Eaton press’. The frozen extracts were stored at -20~. Frozen extract (13 g) was dispersed in z5 ml of distilled water until thawed. Deoxyribonuclease (0.5 ml, I mg/ml) was added and the mixture stirred gently until the viscosity of the solution diminished to that of water. All subsequent procedures were carried out at 4’. The extract was centrifuged once at 13 ooo rev./min for IO min to remove the unbroken cells and cell debris and a second time for 60 min at the same speed to remove other particulate matter. The crude extract was treated with onetenth its volume of 20% streptomycin sulfate to precipitate nucleic acids, centrifuged for IO min to remove the nucleic acid precipitate, and the supernatant solution was dialyzed overnight against distilled water. During this dialysis, a heavy precipitate formed inside the dialysis bag. This precipitate, which contained the enzymatic activity, was recovered by centrifugation, taken up in 20 ml of distilled water and homogenized. Unless otherwide specified, this homogenate was used as the enzyme in these studies. Analytical procedures. Activity of the enzyme was estimated from the release of Pi from ATP. The assay mixture
contained,
in a volume of 0.2 ml, ~-IO-~ M ATP,
0.1 M glutamic acid (pH 7.6), 5.10-~ M MgCl, and 2.5.10-~ M imidazole buffer (pH 7.6) and enzyme. One unit of activity was defined as that amount of enzyme which caused the proline-inhibitable
release of I pmole of Pi per h. Pi was measured
using the reagent of FISKE and SUBBAROW~ after the samples had been deproteinized with 2.5% trichloroacetic acid. Activation of glutamate was estimated by the hydroxamate method of LIP~IANN AND TUTTLE~. Protein was measured by the method of LOWRY et aLlo, using crystalline bovine serum albumin as the standard. RESULTS
A 7.7-fold purification of the enzymatic activity with little loss of activity was achieved (Table I). The release of Pi from ATP requires glutamic acid, MgCl, and imidazole and is inhibited by proline (Table II). In the best preparation, the precipitate obtained upon dialysis after streptomycin treatment, 80 y0 of the release of Pi by the extract was inhibitable by I - IO-~ M proline. Marked substrate activation is observed when the concentration of glutamate exceeds 0.05 M. Below this concentration, a straight line Michealis-Menten curve is obtained with a Km for glutamate of 0.007-0.01 M. The addition of I-IO-~ M proline to the assay mixture increases the apparent Km for glutamic acid, as well as producing B&him.
Biophys.
Acta,
192 (1969) 462-467
A.
464
BAICH
a nonlinear response (Fig. I). Higher concentrations of proline inhibit the reaction almost completely (Fig. 2). Maximum activity of the enzyme was found between pH 7.6 and 8.6 (Fig. 3). At higher pH values. inhibition of the reaction by proline was somewhat overcome. The requirement for imidazole is illustrated in Fig. 4, and low concentrations TABLE
I
PURIFICATION
OF GLUTAMIC
Y-SEMIALDEHYDE
Activity (units)
Crude extract After streptomycin treatment Precipitate after dialysis
TABLE
‘37 151
109
SYNTHETASE
Protein
.SpCCi& activity
(md 2 790
0.0~8
I550
0.097
297
Purification
Recovery (%) IO0
2.0
0.37
7.7
II0
81
II
REQUIREMENT
FOR THE
ENZYMATIC
RELEASE
OF Pi BY
THE
ENZYRlE
The complete system contains, in o.z-ml final volume, 7’ IO-~ M ATP, 0.1 M glutamic acid (pH 7.6), 5’ 10-2 M M&l,, 2,5’ 10-2 M imidazole (pH 7.6) and enzyme. Systenz
Release Strain
Complete + proline (1. ro-3 M) -glutamate -imidazole
IO0 5.9 0 II.8
(% ) Strain
83.7 3.8 16.6
0
0
-M&l,
3.5
0
0
0
I
I
50
OO I.
Biochim.
I
l/S] CM-‘)
100
WPr
100
-enzyme
- ATP
Fig.
of P< 5 j-I
I
150
I
Effect of substrate concentration on velocity of reaction. Samples assayed in the absence resence (A) of I. IO-~ M proline. Concentration of protein was 0.12 mg/o.z ml reaction
Biophys.
Acta,
192
(1969) 462-467
PROLINE
SYNTHESIS
IN
E.
465
COh
Fig. 2. Effect of proline on the velocity volume of reaction mixture.
of reaction.
Concentration
of protein was 0.16 mg per 0.2 ml
Fig. 3, Effect of pH on the velocity of reaction. Samples assayed in the absence (0) and presence (a ) of I. 10-5M proline. Concentration of protein was 0.16 mg/o.a ml volume of reaction mixture.
0 Imidozole
0.0 5
concn.
0.1
CM)
Fig. 4. Effect of imidazole on the velocity of reaction. Samples tested in the absence (0) and presence (a ) of I. IO-~ M proline. Concentration of protein 0.12 mg/o.z ml reaction mixture. TABLE
III
A COMPARISON
OF HYDROXAMIC
ACID
system
Pt released (pmoles)
2 M NH,OH + I. IO-~M proline
7.5 2.5
IO-~
+ I.
M imidazole M proline
IO-~
FORMED
AND
THE
P,
RELEASED
Hydroxamate formed (,umoZes) 6.0
-
5.0 5.
DERIVATIVE
1.0
-
5.0
6.6 I.3 5.3
Biochim. Biophys. Acta, 192 (1969) 462.-467
A. BAICH
466
of proline increase the dependence of the reaction on this compound. Activation of glutamic acid was demonstrated by the formation of a hydroxamic acid derivative. The formation of this derivative was not dependent upon imidazole; the presence of hydroxylamine permits the release of Pr (Table III). DIscussIon:
The rate of the reaction in extracts prepared from a controlled strain of bacteria is a sensitive function of the concentrations of glutamic acid, proline and imidazole. Extracts of strain WPI, which is not subject to end-product inhibition of proline biosynthesis in vivo, catalyzes a corresponding reaction which is less sensitive to the action of proline. The substrate dependence on the rate of reaction shows a cooperative effect only at very high glutamate concentrations under the conditions used here. However, the addition of a low concentration of proline gives the nonlinear Michaelis-Menten curve characteristic of an allosteric effector. Because of the lability of free y-glutamyl phosphaten, an enzyme-bound intermediate is probably involved in the reaction. Two structures may be proposed for this intermediate, y-glutamyl phosphate, which is analogous to products formed in the aspartokinase12 and N-acetylglutamokinase13 reactions, and y-glutamyl enzyme, which is similar to the complex observed in the synthesis of glutamine4. The reaction may occur by way of one or both of these intermediates (Fig. 5). Imidazole and hydroxylamine are considered to cause a nucleophilic displacement of the enzyme from the enzyme-substrate complex14y15. It is not proposed that the imidazole-dependent mechanism operates in vivo but that added imidazole acts as a model or substitute for a subsequent enzyme reaction. The second reaction of the biosynthetic pathway, which catalyzes the formation of glutamic y-semialdehyde, would serve this purpose in vivo. Because the substrate is apparently enzyme (55-1)
Cd0 , ‘OH YNH2
+ATP + Enzyme
tdg2+
_
40
C, I OH
HC-NH2 Enzyme
+
CH2
CH2
CH2 $0, *NOH
Fig. 5. Proposed mechanism for the activation of glutamic acid. Biochim.
Biophys.
Acta,
192 (1969)
462-467
f
Enzyme
PROLINE
SYNTHESIS
IN
E.
467
COli
bound, it could be anticipated that the second enzyme in the pathway is close to, or bound to, the first in whole cells. The reaction described here is implicated in the biosynthesis of proline on the basis of its sensitivity to inhibition by proline, in contrast to glutamine synthetase16, and because extracts of cells which excrete proline in vivo contain a corresponding enzyme which is not inhibited by this compound. ACKNOWLEDGMENTS
This investigation was supported by Public Health Service Grant No. CA-ozzgg, from the National Cancer Institute. I am the recipient of a Research Career Development award from the Public Health Service, National Institute of Allergy and Infectious Diseases. The technical assistance of Mrs. Linda Haley is gratefully acknowledged. REFERENCES I 2 3 4
STRECKER, J. Biol. Chem., 225 (1957) 825. VOGEL, P. H. ABELSON AND E. T. BOLTON, Biochim. Biophys. Acta., II (1953) 584. VOGEL AND B. D. DAVIS, J. Am. Chem. Sot., 74 (1952) Iog. KRISHNASWAMY, V. PAMILJANS AND A. MEISTER, J. Biol. Chem., 237 (1962) 2932. JOHNSTON AND K. BLOCH, J. Biol. Chem., 188 (1951) 221. A. BAICH AND D. J. PIERSON, Biochim. Biophys. Acta., 104 (1965) 397. N. R. EATON, J. Bacteriol., 83 (1962) 1359. C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375. F. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (1945) 21. 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. A. KATCHALSKY AND M. PAECHT, J. Am. Chem. Sot., 76 (1954) 6042. S. BLACK AND N. G. WRIGHT, J. Biol. Chem., 213 (1955) 27. A. BAICH AND H. J. VOGEL, Biochem. Biophys. Res. Commun., 7 (1962) 491. M. L. BENDER AND B. W. TURNQUEST, J. Am. Chem. SOL, 79 (1957) 1652. M. L. BENDER AND B. W. TURNQUEST, J. Am. Chem. Sot., 7g (1957) 1656. T. C. BRUICE AND G. L. SCHMIR, J. Am. Chem. SOL, 79 (1957) 1663. C. A. WOOLFOLK AND E. R. STADTMAN, Biochem. Biophys. Res. Commun., 17 (1964) 313.
H. H. H. P. 5 R. 6 7
8 9 IO II
12 13 14 15 16
17
J. J. J. R. B.
Biochim.
Biophys.
Acta,
192 (1969) 462-467