Hoppe-Seyler's Z. Physiol. Chem. Bd. 356, S. 915-920, Juni 1975
The pH-Dependence of the Peptidase Activity of Aminoacylase Werner Kordel and Friedhelm Schneider
(Received 22 January 1975) Dedicated to Prof. Dr. G. Weitzel on the occasion of his 60 th birthday
Summary: Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveales that only dipeptides with uncharged JV-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the TV-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent.
Hydrophobie, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors, Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the W-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.
Die pH-Abhängigkeit der Peptidaseaktivität der Aminoacylase Zusammenfassung: Aminoacylase besitzt eine hohe Peptidaseaktivität bei pH-Werten um 8.5. Die pH-Abhängigkeit der Km -Werte verschiedener Peptids zeigt, daß nur Dipeptide mit ungeladener Af-terminaler Aminosäure vom Enzym gebunden werden. Die Km -Werte sind um so niedriger, je hydrophober die Af-terminale Aminosäure ist und fallen in der Reihenfolge Thr > Gly > Ala > Leu. Die Km -Werte werden pH-unabhängig, wenn man sie auf der Basis der Konzentration der deprotonierten Peptide berechnet.
Inhibitorkonstanten mit Glycylalanin als Substrat betragen für Tryptophan 1.2 10~4 , Norleucin 1.2 10~ 4 M, Leucin4.8 10~ 4 M, Valin 18 10~ 4 M, Phenylalanin 21 10~4 und Isoleucin 34.8 10~4 . Die Größe der Inhibitorkonstante wird bestimmt durch die Hydrophobizität der Aminosäure sowie sterische Faktoren. Aus den Befunden ist zu schließen, daß die Bindung der N-terminalen Aminosäure der Dipeptide an einem streng hydrophoben Zentrum erfolgt.
Hydrophobe, deprotonierte Aminosäuren sind starke kompetitive Inhibitoren des Enzyms.
Address: Prof. Dr. Fr. Schneider, Physiologisch.-Chem. Institut II, Marburg Lahnberge. Enzyme: Aminoacylase, 7V-acylamino-acid amidohydrolase (EC 3.5.1.14).
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916
W. Kordel and F. Schneider
Bd. 356(1975)
Fig. 1. Lineweaver-Burk plots for the hydrolysis of N-chloroacetylalanine catalyzed by aminoacylase at different pH values (figures at the curves). -150
Ο
Aminoacylase catalyzes the hydrolysis of acylamino acids^1"3' and, near pH 7 at much lower rates, the splitting of dipeptidesl2'4'. Nothing is known about the physiological significance and the catalytic mechanism of the enzyme. In the course of our studies on the molecular mechanism of amide hydrolysis we have investigated the pH dependence of the peptidase activity of aminoacylase to obtain information on the character of the binding center of the enzyme. In the present communication we report the results of these experiments.
The Henderson-Hasselbach equation and the pK values of the peptides were used for the calculation of the concentration of deprotonated peptides at different pH values. Inhibitor constants of amino acids were determined according to Dixoni 7 l
Materials and Methods Aminoacylase was a gift of Boehringer Mannheim GmbH. The enzyme was purified by chromatography on aminoagarose columns's I The purified enzyme was homogeneous as judged by disc electrophoresis. Activity measurements were performed spectrophotometrically with a Zeiss photometer PMQII at 40 °C. Hydrolysis of ,/V-chloroacetylalanine was folio wed at 238 nm and the hydrolysis of dipeptides at 235 nm. All measurements were carried out in 0. IM phosphate/ borate buffer. pK Values of the dipeptides at 40 °C were determined by potentiometric titration with a Radiometer Autotitrator arrangement. Chloroacetylalanine was synthesized according to refJ 6 J, peptides were purchased from Fluka, Buchs.
Fig. 2. pH Dependence of the activity of aminoacylase with Chloroacetylalanine, glycylalanine and deprotonated glycylalanine.
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Bd. 356 (1975)
917
The pH-Dependence of the Peptidase Activity of Aminoacylase
1200
I/[SI ft/mlJ-
Results and Discussion The pH dependence of the hydrolysis of Λ^-chloroacetylalanine catalyzed by aminoacylase is demonstrated in Fig. 1 and 2. The Lineweaver-Burk plots of Fig. 1 reveal a pH independent apparent Km of 6.6 χ 10~ 3 M for this acylamino acid. The pH optimum of the peptidase activity with glycylalanine as substrate is shifted to higher pH values compared to the acylase activity. If we calculate the pH-dependence of the hydrolysis of glycylalanine on the basis of the concentration of unprotonated peptide we get the intermediate eilfve of Fig. 2. The pH optimum of peptidase activity is now shifted in the direction of acylase activity. Table 1. £ m app at pH 8.6, pH-independent A"mapp and maximal velocity K for the hydrolysis of dipeptides catalyzed by aminoacylase.
Dipeptide
As is demonstrated in Fig. 3 with glycyl-leucine as substrate the apparent Km for dipeptides drops with increasing pH in contrast to the Km values of acylamino acids. If one expresses the apparent Km in terms of the concentration of the deprotonated peptides then they become pH-independent as is the case with chloroacetylalanine. The values of Km app at pH 8.6 and calculated pH-independent Km app as well as the maximum velocity for a series of dipeptides are summarized in Table 1. These observations suggest that only peptides with unprotonated amino groups are the real substrates of aminoacylase. From Table 1 it is further evident that the enzyme has an appreciable peptidase activity at pH 8.6; especially good
10 3 xK m [moll I]
Gly-Gly Gly-Ala Gly-Leu Gly-Ser Ala-Leu Leu- Leu Thr-Ala Leu-Ala ClCH2COAla
Fig. 3. Lineweaver-Burk plots for the hydrolysis of glycylleucine catalyzed by aminoacylase at different pH values.
7.1 6.6 6.6 6.6 5.0 0.25 15.0 1.8 6.6
pH-independent 10 3 x* m [mol//]
[nmol/(/ χ min x mg prot.J
5.8 4.9 5.1 5.0 4.0 0.20 14.2 1.5 6.6
4.5 ' 27.7 55.5 21.7 62.5 2.4 7.1 2.0 250.0
V
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918
W. Kordel and F. Schneider
Bd. 356(1975)
100-
Leu fmmol/l) -
Leu.r (mmol/lj-
Fig.4 a) pH-dependence of the inhibition of aminoacylase by leucine. Substrate: chloroacetylalanine. pH values as indicated at the curves. b) pH-Independent inhibition of aminoacylase by deprotonated leucine.
substrates being glycyl-leucine and alanyl-leucine. Publications to date have only described the peptidase activity observed near neutralityi^4'. Aminoacylase was therefore considered to be only a poor peptidase. Further arguments for the deprotonated peptides being the true substrates stem from inhibition experiments. Amino acids are competitive inhibitors of aminoacylase. The pH dependence of the inhibition of the hydrolysis of chloroacetylalanine by leucine is shown in Fig. 4 a. This diagram reveals an increasing inhibition by leucine with increasing pH. Expressing the inhibition in terms of the concentration of the deprotonated amino acid one gets Fig. 4 b. This figure represents the concentration dependence of the pH-independent inhibition of aminoacylase by leucine. The inhibition of the peptidase activity of aminoacylase by different amino acids in terms of the K{ values is summarized in Table 2, which also contains the hydrophobicity parameters for the side chains according to Tanfordl8!. The relation between the inhibitor constant and the hydrophobicity of the respective amino acids is illustrated in Fig. 5. From this graph it becomes evident that the inhibitory effects of the amino acids are first of
?
*
AF[kJ/mol] —»B 1
ψ
γ
Leu
Abu/ j A
1
t~
/A«,u
ψ
Phe He
/l/a/'
4? 2
AFfkcal/mol] —*~
3
Fig. 5. Relation between inhibitor constants and hydrophobicity parameters of the amino acid side chains.
all determined by the hydrophobic character of the amino acid. The following further conclusions may be drawn from our results. The strongest inhibitors are straight chain amino acids, their KI values rising with increasing chain length. Branching of the side chains reduces the binding of the amino acids by the enzyme. This effect is made especially evident by comparing norleu-
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The pH-Dependence of the Peptidase Activity of Aminoacylase
Bd. 356(1975)
919
Table 2. Inhibitor constant of different amino acids for the inhibition of the hydrolysis of glycylalanine catalyzed by aminoacylase and hydrophobicity parameters of amino acid side chains. ΙΟ4 χ Ki
Amino acid
P*i
[mol//]* Tryptophan Norleucine Norvaline α-Aminobutyric acid a-Aminoisobutyric acid Valine Phenylalanine Alanine Isoleucine Glutamine Glycine Threonine
1.2 1.2 1.3 6.0 12.0 18.0 21.0 32.0 35.0 139.0 180.0 223.0
3.91 3.90 3.87 3.22 2.92 2.74 2.67 2.49 2.47 1.86 1.74 1.65
Δ/τ** kJ mol
rkcall LmolJ
12.55 11.30 9.16 6.03 5.36*** 7.07 11.09 3.05 12.43 -0.4 0 1.84
3.0 2.7 2.19 1.44 1.28*** 1.69 2.65 0.73 2.97 -0.1 0 0.44
* Calculated on the basis of the deprotonated L-amino acids. ** Side chain contribution of free energy for transfer of the amino acids from ethanol to water according to Tanfordl8!. *** The AFvalue was calculated from the value for alanine and an increment for a secondary methyl group of 2.30 kJ (= 0.55 kcal)/mol(8].
cations that it is a tryptophan of the binding site. This observation is in accord with the well known participation of tryptophan residues in substrate 9 Obviously these amino acids do not fit the binding binding and their role in determining specificity( K center for steric and conformational reasons; that The following conclusions may be drawn from is also the case for phenylalanine. The.effect of our experiments. The acyl moiety of the acylintroducing a hydrophilic group into the side amino acids and the W-terminal amino acid of chain is well demonstrated by the low inhibition the dipeptides are bound to a strongly hydroconstant of threonine compared to a-aminophobic center. From Table 1 one recognizes that butyric acid. Negative charges in the side chain Km app decreases in the following sequence: (glutamic acid) prevent any interaction with the Thr > Gly > Ala > Leu. The rate of hydrolysis binding center as do positive charges. rises from threonine via glycine to alanine but drops considerably with leucine. This is a conseA special position is occupied by tryptophan quence of the strong binding of leucine which which is the strongest competitive inhibitor of leaves the hydrophobic binding center at a low the enzyme besides norleucine. This result is rate. somewhat surprising and indicates an especially strong interaction of the binding site with The influence of the second amino'acid on the tryptophan. At this point it must be mentioned rate of hydrolysis of dipeptides is the same as that highly specific chemical modification of described by Greensteinl2! in studies on the hydroone tryptophan residue abolishes the catalytic lysis of acylamino acids catalyzed by aminoactivity of the enyme*. There are several indiacylase.
cine, leucine and isoleucine or norvaline and valine or a-aminobutyric acid and a-aminoisobutyric acid.
* Kordel, W. & Schneider, F., unpubl. results.
The experiments were supported by a grant from Deutsche Forschungsgemeinschaft.
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920
W. Kordel and F. Schneider
Bd. 356(1975)
Literature 1 Schmiedeberg, O. (1881) Naunyn-Schmiedebergs Archiv Exp. Pathol. Pharmakol. 13, 379 - 392. 2 Rao, K.R., Birnbaum, S.M., Kingsley, R.B. & Greenstein, LP. (1952) /. Biol. Chem. 198, 507 - 523. 3 Ötvos, L., Moravcsik.E. & Mady, G. (1971) Biochem. Biophys. Res. Commun. 44, 1056 -1063. 4 Moravcsik, E., Tüdös, H., Telegdy, J., Kömives, K. & Ötvos, L. (1974) 9thFEBS Meeting, Budapest, Abstracts p. 91.
5
Kordel, W. (1974) Biochem. Diplomarbeit, Marburg. Birnbaum, S.M., Levintow, L., Kingsley, R.B. & Greenstein, I. P. (1952) J. Biol. Chem. 194, 455 - 470. 7 Dixon, M. (1953) Biochem. J. 55, 170 - 171. 8 Tanford, C. (1962) J. Amer. Chem. Soc. 84, 4240 - 4247. 9 Vallee, B.L. & Riordan, J.F. (1969) Annu. Rev. Biochem. 38, 733 - 794. 6
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The pH-Dependence of the Peptidase Activity of Aminoacylase Werner Kordel and Friedhelm Schneider
(Received 22 January 1975) Dedicated to Prof. Dr. G. Weitzel on the occasion of his 60 th birthday
Summary: Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveales that only dipeptides with uncharged JV-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the TV-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent.
Hydrophobie, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors, Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the W-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.
Die pH-Abhängigkeit der Peptidaseaktivität der Aminoacylase Zusammenfassung: Aminoacylase besitzt eine hohe Peptidaseaktivität bei pH-Werten um 8.5. Die pH-Abhängigkeit der Km -Werte verschiedener Peptids zeigt, daß nur Dipeptide mit ungeladener Af-terminaler Aminosäure vom Enzym gebunden werden. Die Km -Werte sind um so niedriger, je hydrophober die Af-terminale Aminosäure ist und fallen in der Reihenfolge Thr > Gly > Ala > Leu. Die Km -Werte werden pH-unabhängig, wenn man sie auf der Basis der Konzentration der deprotonierten Peptide berechnet.
Inhibitorkonstanten mit Glycylalanin als Substrat betragen für Tryptophan 1.2 10~4 , Norleucin 1.2 10~ 4 M, Leucin4.8 10~ 4 M, Valin 18 10~ 4 M, Phenylalanin 21 10~4 und Isoleucin 34.8 10~4 . Die Größe der Inhibitorkonstante wird bestimmt durch die Hydrophobizität der Aminosäure sowie sterische Faktoren. Aus den Befunden ist zu schließen, daß die Bindung der N-terminalen Aminosäure der Dipeptide an einem streng hydrophoben Zentrum erfolgt.
Hydrophobe, deprotonierte Aminosäuren sind starke kompetitive Inhibitoren des Enzyms.
Address: Prof. Dr. Fr. Schneider, Physiologisch.-Chem. Institut II, Marburg Lahnberge. Enzyme: Aminoacylase, 7V-acylamino-acid amidohydrolase (EC 3.5.1.14).
Brought to you by | Université Paris Ouest Nanterre La Défense Authenticated Download Date | 12/11/16 8:38 PM
916
W. Kordel and F. Schneider
Bd. 356(1975)
Fig. 1. Lineweaver-Burk plots for the hydrolysis of N-chloroacetylalanine catalyzed by aminoacylase at different pH values (figures at the curves). -150
Ο
Aminoacylase catalyzes the hydrolysis of acylamino acids^1"3' and, near pH 7 at much lower rates, the splitting of dipeptidesl2'4'. Nothing is known about the physiological significance and the catalytic mechanism of the enzyme. In the course of our studies on the molecular mechanism of amide hydrolysis we have investigated the pH dependence of the peptidase activity of aminoacylase to obtain information on the character of the binding center of the enzyme. In the present communication we report the results of these experiments.
The Henderson-Hasselbach equation and the pK values of the peptides were used for the calculation of the concentration of deprotonated peptides at different pH values. Inhibitor constants of amino acids were determined according to Dixoni 7 l
Materials and Methods Aminoacylase was a gift of Boehringer Mannheim GmbH. The enzyme was purified by chromatography on aminoagarose columns's I The purified enzyme was homogeneous as judged by disc electrophoresis. Activity measurements were performed spectrophotometrically with a Zeiss photometer PMQII at 40 °C. Hydrolysis of ,/V-chloroacetylalanine was folio wed at 238 nm and the hydrolysis of dipeptides at 235 nm. All measurements were carried out in 0. IM phosphate/ borate buffer. pK Values of the dipeptides at 40 °C were determined by potentiometric titration with a Radiometer Autotitrator arrangement. Chloroacetylalanine was synthesized according to refJ 6 J, peptides were purchased from Fluka, Buchs.
Fig. 2. pH Dependence of the activity of aminoacylase with Chloroacetylalanine, glycylalanine and deprotonated glycylalanine.
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Bd. 356 (1975)
917
The pH-Dependence of the Peptidase Activity of Aminoacylase
1200
I/[SI ft/mlJ-
Results and Discussion The pH dependence of the hydrolysis of Λ^-chloroacetylalanine catalyzed by aminoacylase is demonstrated in Fig. 1 and 2. The Lineweaver-Burk plots of Fig. 1 reveal a pH independent apparent Km of 6.6 χ 10~ 3 M for this acylamino acid. The pH optimum of the peptidase activity with glycylalanine as substrate is shifted to higher pH values compared to the acylase activity. If we calculate the pH-dependence of the hydrolysis of glycylalanine on the basis of the concentration of unprotonated peptide we get the intermediate eilfve of Fig. 2. The pH optimum of peptidase activity is now shifted in the direction of acylase activity. Table 1. £ m app at pH 8.6, pH-independent A"mapp and maximal velocity K for the hydrolysis of dipeptides catalyzed by aminoacylase.
Dipeptide
As is demonstrated in Fig. 3 with glycyl-leucine as substrate the apparent Km for dipeptides drops with increasing pH in contrast to the Km values of acylamino acids. If one expresses the apparent Km in terms of the concentration of the deprotonated peptides then they become pH-independent as is the case with chloroacetylalanine. The values of Km app at pH 8.6 and calculated pH-independent Km app as well as the maximum velocity for a series of dipeptides are summarized in Table 1. These observations suggest that only peptides with unprotonated amino groups are the real substrates of aminoacylase. From Table 1 it is further evident that the enzyme has an appreciable peptidase activity at pH 8.6; especially good
10 3 xK m [moll I]
Gly-Gly Gly-Ala Gly-Leu Gly-Ser Ala-Leu Leu- Leu Thr-Ala Leu-Ala ClCH2COAla
Fig. 3. Lineweaver-Burk plots for the hydrolysis of glycylleucine catalyzed by aminoacylase at different pH values.
7.1 6.6 6.6 6.6 5.0 0.25 15.0 1.8 6.6
pH-independent 10 3 x* m [mol//]
[nmol/(/ χ min x mg prot.J
5.8 4.9 5.1 5.0 4.0 0.20 14.2 1.5 6.6
4.5 ' 27.7 55.5 21.7 62.5 2.4 7.1 2.0 250.0
V
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918
W. Kordel and F. Schneider
Bd. 356(1975)
100-
Leu fmmol/l) -
Leu.r (mmol/lj-
Fig.4 a) pH-dependence of the inhibition of aminoacylase by leucine. Substrate: chloroacetylalanine. pH values as indicated at the curves. b) pH-Independent inhibition of aminoacylase by deprotonated leucine.
substrates being glycyl-leucine and alanyl-leucine. Publications to date have only described the peptidase activity observed near neutralityi^4'. Aminoacylase was therefore considered to be only a poor peptidase. Further arguments for the deprotonated peptides being the true substrates stem from inhibition experiments. Amino acids are competitive inhibitors of aminoacylase. The pH dependence of the inhibition of the hydrolysis of chloroacetylalanine by leucine is shown in Fig. 4 a. This diagram reveals an increasing inhibition by leucine with increasing pH. Expressing the inhibition in terms of the concentration of the deprotonated amino acid one gets Fig. 4 b. This figure represents the concentration dependence of the pH-independent inhibition of aminoacylase by leucine. The inhibition of the peptidase activity of aminoacylase by different amino acids in terms of the K{ values is summarized in Table 2, which also contains the hydrophobicity parameters for the side chains according to Tanfordl8!. The relation between the inhibitor constant and the hydrophobicity of the respective amino acids is illustrated in Fig. 5. From this graph it becomes evident that the inhibitory effects of the amino acids are first of
?
*
AF[kJ/mol] —»B 1
ψ
γ
Leu
Abu/ j A
1
t~
/A«,u
ψ
Phe He
/l/a/'
4? 2
AFfkcal/mol] —*~
3
Fig. 5. Relation between inhibitor constants and hydrophobicity parameters of the amino acid side chains.
all determined by the hydrophobic character of the amino acid. The following further conclusions may be drawn from our results. The strongest inhibitors are straight chain amino acids, their KI values rising with increasing chain length. Branching of the side chains reduces the binding of the amino acids by the enzyme. This effect is made especially evident by comparing norleu-
Brought to you by | Université Paris Ouest Nanterre La Défense Authenticated Download Date | 12/11/16 8:38 PM
The pH-Dependence of the Peptidase Activity of Aminoacylase
Bd. 356(1975)
919
Table 2. Inhibitor constant of different amino acids for the inhibition of the hydrolysis of glycylalanine catalyzed by aminoacylase and hydrophobicity parameters of amino acid side chains. ΙΟ4 χ Ki
Amino acid
P*i
[mol//]* Tryptophan Norleucine Norvaline α-Aminobutyric acid a-Aminoisobutyric acid Valine Phenylalanine Alanine Isoleucine Glutamine Glycine Threonine
1.2 1.2 1.3 6.0 12.0 18.0 21.0 32.0 35.0 139.0 180.0 223.0
3.91 3.90 3.87 3.22 2.92 2.74 2.67 2.49 2.47 1.86 1.74 1.65
Δ/τ** kJ mol
rkcall LmolJ
12.55 11.30 9.16 6.03 5.36*** 7.07 11.09 3.05 12.43 -0.4 0 1.84
3.0 2.7 2.19 1.44 1.28*** 1.69 2.65 0.73 2.97 -0.1 0 0.44
* Calculated on the basis of the deprotonated L-amino acids. ** Side chain contribution of free energy for transfer of the amino acids from ethanol to water according to Tanfordl8!. *** The AFvalue was calculated from the value for alanine and an increment for a secondary methyl group of 2.30 kJ (= 0.55 kcal)/mol(8].
cations that it is a tryptophan of the binding site. This observation is in accord with the well known participation of tryptophan residues in substrate 9 Obviously these amino acids do not fit the binding binding and their role in determining specificity( K center for steric and conformational reasons; that The following conclusions may be drawn from is also the case for phenylalanine. The.effect of our experiments. The acyl moiety of the acylintroducing a hydrophilic group into the side amino acids and the W-terminal amino acid of chain is well demonstrated by the low inhibition the dipeptides are bound to a strongly hydroconstant of threonine compared to a-aminophobic center. From Table 1 one recognizes that butyric acid. Negative charges in the side chain Km app decreases in the following sequence: (glutamic acid) prevent any interaction with the Thr > Gly > Ala > Leu. The rate of hydrolysis binding center as do positive charges. rises from threonine via glycine to alanine but drops considerably with leucine. This is a conseA special position is occupied by tryptophan quence of the strong binding of leucine which which is the strongest competitive inhibitor of leaves the hydrophobic binding center at a low the enzyme besides norleucine. This result is rate. somewhat surprising and indicates an especially strong interaction of the binding site with The influence of the second amino'acid on the tryptophan. At this point it must be mentioned rate of hydrolysis of dipeptides is the same as that highly specific chemical modification of described by Greensteinl2! in studies on the hydroone tryptophan residue abolishes the catalytic lysis of acylamino acids catalyzed by aminoactivity of the enyme*. There are several indiacylase.
cine, leucine and isoleucine or norvaline and valine or a-aminobutyric acid and a-aminoisobutyric acid.
* Kordel, W. & Schneider, F., unpubl. results.
The experiments were supported by a grant from Deutsche Forschungsgemeinschaft.
Brought to you by | Université Paris Ouest Nanterre La Défense Authenticated Download Date | 12/11/16 8:38 PM
920
W. Kordel and F. Schneider
Bd. 356(1975)
Literature 1 Schmiedeberg, O. (1881) Naunyn-Schmiedebergs Archiv Exp. Pathol. Pharmakol. 13, 379 - 392. 2 Rao, K.R., Birnbaum, S.M., Kingsley, R.B. & Greenstein, LP. (1952) /. Biol. Chem. 198, 507 - 523. 3 Ötvos, L., Moravcsik.E. & Mady, G. (1971) Biochem. Biophys. Res. Commun. 44, 1056 -1063. 4 Moravcsik, E., Tüdös, H., Telegdy, J., Kömives, K. & Ötvos, L. (1974) 9thFEBS Meeting, Budapest, Abstracts p. 91.
5
Kordel, W. (1974) Biochem. Diplomarbeit, Marburg. Birnbaum, S.M., Levintow, L., Kingsley, R.B. & Greenstein, I. P. (1952) J. Biol. Chem. 194, 455 - 470. 7 Dixon, M. (1953) Biochem. J. 55, 170 - 171. 8 Tanford, C. (1962) J. Amer. Chem. Soc. 84, 4240 - 4247. 9 Vallee, B.L. & Riordan, J.F. (1969) Annu. Rev. Biochem. 38, 733 - 794. 6
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