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Investigations on the Antiproliferative Effects of Amino Acid Antagonists Targeting for Aminoacyl-tRNA Synthetases Part I - The Antibacterial Effect.

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847
AntiproliferativeEffects of Amino Acid Antagonists
Investigations on the Antiproliferative Effects of Amino Acid
Antagonists Targeting for Aminoacyl-tRNA Synthetases
-
Part I The Antibacterial Effect
Reiner Laske' and Helmut Schonenberger
Institut f i r Pharmazie der Universitiit Regensburg, Universitiitsstr.3 1, D-8400 Regensburg, FRG
Eggehard Holler
Institut fiir Biophysik und physikalische Biochemie der Universitiit Regensburg, Universitiitsstr.31. D-8400 Regensburg, FRG
Received November 24,1988
Amino acid antagonists with proven or potentially inhibitory activities on
aminoacyl-tRNA synthetases were tested for their antiproliferative effect
against E.coli B. The compounds4- and 6-fluomtryptophan, 5-methyltryptophan, selenocystineand 6-(2-thienyl)alaninegave smng growth inhibition in
minimal medium, which disappeared after addition of structurally related
natural amino acids or in an enriched broth. The inhibitory effect on aminoacyl-tRNA synthetases and the minimal inhibitory concentration for growth
inhibitionin minimal medium could not be correlated.
Protein biosynthesis seems to be M ideal target for antagonists of all 20
physiological amino acids in order to produce antiproliferative effects as
part of a chemotherapy of bacterial') or malignant4g)diseases. In this
respect, aminoacyl-tRNA synthetases(AARS)2) are an interesting and relevant group of target enzymes. AARS catalyze the chemical activation and
the transfer of natural amino acids (AA) to their cognate tRNAAA's.
-
MgATpz' + Amino Acid + AARSAA c'AARSAA.Aminoacyl AMP +
MPP2Q20)
AARSAA.Aminoacyl-AMP
+ tRNAAAt' AARSAA
Aminoacyl-tRNA
+ AMP
(ii)
After dissociation from AARS the aminoacyl-tRNA delivers its amino
acid in a chemically activated state to the ribosomal protein synthesis apparatus, where don-anticodon base pairing between mRNA and tRNA accomplishes the translation of the nucleic acid code into an amino acid sequence of a protein. A crucial role of A A R S is the esterification of the
comct amino acid with the cognate tRNA in order to guarantee the accuracy of translation of the genetic code. This is a achieved by the existence of
at least one specific AARS per amino acid and by a 2-step recognition
DrOcess for the amino acid at the level of the soecific AARS. In the first
step, the binding to a specific AARS is obtained by the sterical and chemic d properties of the side chain of the zwitterionic amino acid. In a second
step, the enzyme performs a proofreading or editing of the formed
Arch. Pharm. (Weinheim)322,847-8S2 (1989)
Untersuchungen mr antiproliferativen Wirkung von Aminosiiureantagonisten mit dem Target Aminoacyl-tRNA-Synthetasen.
Teil I Antibakterieller Effekt
-
Aminosiiureantagonisten mit erwiesener oder potentieller Hemmwirkung an
Aminoacyl-tRNA-Synthetasen wurden auf ihre antiproliferativen Eigenschaften an E.coli B untersucht. Die Verbindungen 4- bzw. 6-Fluortryptophan, 5-Methyltryptophan, Selenocystin und 8-(2-Thienylalanin) enielten
bereits in submikromolaren Konzentrationen auf Minimalniihrboden einen
guten wachstumshe-mmenden Effekf diese Wirkung verschwand aber bei
Zusatz strukturvewandter natUrlicher Aminosbren bzw. bei Testungen in
Vollmedium Zwischen der Hemmwirkung an Aminoacyl-tRNA-Synthetasen und der minimalen Hemmkomntmtion in Minimalmedium konnte
kine Korrelation festgestelltwerden
aminoacyl adenylate and/or aminoacyl-tRNA verified by hydrolysis of the
misactivated/misacylated product3'.
Amino acid antagonists can be divided into three groups according to the
mechanism, by which they inhibit the synthesis of functional proteins:
group 1 acts by competitive inhibition [especially by compounds without
carboxylic groups that cannot be chemically activated, e.g. histidinol
(31)4']. Group 2 acts by gentile activation, i.e. the inhibitor is activated by
AARS, but then released by proofreading. These compounds can reenter
the activation and hydrolysis circle resulting in futile enzyme activity and
in permanent ATPconsumption. Also the synthesis of the putative cell
growth activator adenosine (5') tetraphospho (S')-adenosine (AP~A)~'
is inhibited by groups 1 and 2. Because of depletion of the cellular pool, all
aminoacyl-tRNA dependent processes are affected6). Group 3 of amino
acid antagonists act by misacylation; they mimic the natural amino acid so
perfectly that they are activated, wansferred to tRNA and released as
aminoacyl-tRNA. They can thus be incorporated into proteins, which can
result in the formation of nonfunctional proteins? [e.g. Cfluorotryptophan
(95)or azetidine-2-carboxylicacid (83)*'1.
Differences in substrate specifities and proof-reading mechanisms
between eukaryotic and bacterial A A R S could enable such groups of
amino acid antagonists to a selective inhibition of the bacterial enzymes
causing a stop of protein synthesis and proliferation').
In the present work, the antiproliferative effect against
E.coli B of amino acid derivatives as AARS-inhibitors was
investigated. It was Of interest, whether potent growth inhibitors that antagonize amino acids act by mechanisms outlined above.
OVCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1989
0365-6233/89/1212-0847$02.50/0
848
Laske, SchBnenberger, and Holler
Results
After evaluation of the relevant literature out of about 800
described substances with inhibitory activity against AARS
the most interesting compounds as well as substances structurally related to them were chosen (Table 1).
In a first experiment, all compounds were screened for
their antiproliferative effect against E.coli B in a glucosesalt-medium (minimal medium, MM) at an initial concentration of 256 pglml. The absence of amino acids in this medium allowed the analogues to express their maximal antimetabolic effect. In this system 42 of the 121 tested compounds showed an antiproliferative effect [minimal inhibitory concentration (MIC) < ImM]. Table 2 summarizes those
compounds, which were active at concentrations below
1mM. The most active ones were 17, 81, 95, 96, and 103
with a MIC below 1pM. All these compounds except 103
belonged to the third group of inhibitors (incorporated into
protein) and showed strong to moderate enzyme inhibition.
As was expected in medium with high, non-specified concentrations of amino acids (Miiller-Hinton-broth, MHB), a
marked reduction in the antiproliferative effect was observed in accord with the antimetabolic mode of action.
Only 5 compounds gave a MIC below 1mM: 17 (383 pM),
95 and 96 (576 pM), 59 (244
and 63 (228 pM)(Table
2). The effect of the first three compounds was reduced by a
factor of 1/1000 compared with their MIC in MM. Considering 63 and 59, there was a much smaller reduction of
the antibacterial effect in MHB indicating either a very
strong antagonism or no competition with broth ingredients.
In order to confirm an amino acid antagonistic effect, the
most effective compounds were examined in competition
tests by adding their respective amino acids to MM. Their
antibacterial effect could be neutralized each by one or two
natural amino acids. An inhibition index” (ratio of the concentration of the antagonist to the concentration of the agonistic amino acid that was just necessary for inhibition of
growth, II) was determined. 16,17,63,91, and 32 exhibited
w)
Table 1:Tested compounds arranged according to their structurallyrelated (antagonized)natural amino acids
Amino Acid
Antagonist
Alanine:
Arginine:
Asparagine:
L-Alanine methyl ester. HCI (l), L-6-chloroalanine (2). 2-aminoisobutyricacid (3).3,3,3-trifluoro-DL-alanine(4)
L-canavanine [(2-amino-4-(guanidinooxy)butyricacid] (5)
L-2-amino-3-sulfamoylpropionicacid (6), L-aspartic acid diamide . HCI (7). L-aspartic acid-6-hydroxamate (8). DL-asparagine
benzyl ester (9), 6-cyano-L-alanine(10). L-isoasparagine(11)
aminomalonic acid diethyi ester (12), DL-3-aminobutyric acid (13), meso-2,3-diaminosuccinicacid (14)
cysteamine . HCI (15), selenocystamine(16),DL-selenocystine (17). S-trityl-L-cysteine(18)
albizziin (L-2-amino-3-ureidopropionicacid) (19), Camino-n-butyric acid amide . HCI (20) S-carbamyl-DL-cysteine (21), DL-5-hydroxylysine . HCI (22), L-lysinhydroxamate HCI (23). 3-(N-phenylacetyl)amino-2,6-piperidinedione(antineoplastonA10) (24)
4-aminobutyric acid (25).4-amino-4-phosphonobutyric
acid (26). ethionamide (27)
N-CBZglycine (ZS),N-phenylglycine(29)
1,2-diamino-3-(4-imidazolyl)propane~3HCI (histidinamine)(30), L-histidinol(31). a-methylhistidine (32)
L-isoleucinol(33). (S)-2-1nethylbutylamine (34).LO-methylthreonine (39, DL-4-thiaisoleucine (36)
DLannentomycin (2-amino-4,4-dichlorobutyricacid) (37), DL4azaleucine . 2HC1 (38),DL-3-dehydroarmentomycin(39), DL-3hydroxyleucine (40). L-leucinamide . HCI (41). L-leucinol(42), 5,5,5-trifluoro-DL-leucine(43)
DL-a-amino*-caprolactam (44). 6-(3-aminocyclohexyl)-DL-aIanine. HCI (45). DL-pchloroamphetamine (46), trans-2,6diaminoacid methyl ester (48).DL-5-hydroxylysine . HCI (21), L-lysinhydroxamahex-4-enoic acid . HCI (47). DL-2,6-diphthalimidocaproic
te . HCI (23). DL4oxalysine. 2HC1(49), DL4selenalysine . HCI (SO),L4thialysine (51)
DL-ethionine (52). L-methioninol(53). L-methioninamide . HCI (54). a-methyl-DL-methionine(55),seleno-Dlmethionine (56)
2-amino-4-methylhex-4-enoicacid (57). (1S,2S)-2-amino- I-phenyl-l,3-propanediol(58), N-benzyl-D-amphetamine HCI (59). Nbenzyl-L-phenylalanine (60)). N-benzyl-D-phenylalaninol. HCI (6l), N-benzyl-2-phenylethylamine. HCI (62), 1,3-bis(acetoxy)-2nitro-1-phenylpropane (fenitropan) (63). 1.2-diamino-3-(2.6-dichIorophenyl)propane . HCI (64). 1,2-diamino-3-hydroxy-5-phenylpentane (65). 1,2-diamin0-3-phenylpropane(66). N,N’-di-CBZ-L-lysine (67). N-(2,6-dichlorobenzylidene)-2-phenylethylamine(68).
N-(2,6-dichlorobenzyI)-2-phenylethylamine . HCI (69), N-(4-fluorobenzyl)-L-phenylalanine (70). DL-2-fluorophenylalanine
(71),DL-3-fluorophenylalanine( 7 9 , DL4fluorophenylalanine (73), 2-hydroxyethyl-2-phenylammoniumsulfate (74), a-methyl-DLphenylalanine (75),6-methyl-DL-phenylalanine
. HCI (76), L-mimosine [3-(3-hydroxy-4-0xo-l (4H)-pyridyl)-L-alanine](73, L-phenylalaninol(78), La-phenylglycine (79), DL-Ilueo-B-phenylserine (SO), 6-2-thienyl-DL-alanine (81). N-trifluoroacetyl-L-phenylalanine cyclohexyl ester (82)
L-azetidine-2-carboxylicacid (83). DL-3.4-dehydroproline (84),2-aminomethyl-4-isopmpyloxypyrrolidineoxalate (S),2-aminomethylpyrrolidine .2HC1(86), L-4-thiaproline(87)
N-benzylethanolamine (88).N-(2,6-dicNorobenzyl)ethanolamine(89). N-(2.6-dichlorobenzylidene)ethanolamine(90). DL-serine hydroxamate (91)
DL-P-hydroxynorvaline (92), DL-P-hydroxyleucine (40), 1,2-diamino-5-phenyl-3-pentanol(93)
DL-7-azanyptophan (94), DL-4-fluorotryptophan (95). DL-6-fluorotryptophan(96), 5-hydroxytryptamine. HCI (m,L-5-hydroxytryptophan (98). DL-a-methyltryptamine (99). a-methyl-DL-tryptophan (100). 6-methyl-DL-tryptophan(101). 1-methyl-DL-tryptophan (102). 5-methyl-DL-tryptophan(103), 7-methyl-DL-tryptophan(104), Nu-methyl-L-tryptophan(105). tryptamine . HCI (106).
L-tryptophanhydroxamate (107)
DL-2-amino-l-(4-hydroxyphenyl)-l-propanol
. HCI (108). DL-3,4-dihydmxyphenylahine (DOPA) (109), DL-3-fluorotyrosine
(110). 3-hydroxytyramine . HCI (dopamine) ( l l l ) , 3-iodo-L-tyrosine(112), 3-nitro-L-tyrosine(113),tyramine . HCI (114). Ltyosino1 . HCI (115)
S-adenosyl-L-homocysteine(116), L-threo-2-amino-3-chlorobutyric
acid (117). N-CBZ-L-valine (118). 4,4,4,5,5,5-hexafluoro-DL
valine (119). DLnorvaline (120)). L-penicillamine(6-mercaptovaline)(121)
Aspartic Acid
Cysteine:
Glutamine:
9
Glutamic Acid:
Glycine:
Histidine:
Isoleucine:
Leucine:
Lysine:
Methionine:
Phenylalanine:
Proline:
Serine:
Threonine:
Tryptophan:
Tyrosine:
Valine:
1
a
Arch. Pharm. (Weinheim) 322, 847-852 (1989)
849
Antiproliferative Effects of Amino Acid Antagonists
Table 2: List of active amino acids antaeonists and their inhibitorv effects on AARS and proliferation of E.coli
Amino Acid
Antagonist
AARS
Ki/Kma
MIC (PM)
MM*
MHB'
I1
MM~
~
Alanine
2
13
> 1600
Arginine
5
11
2900
Asparagine
8
27
> 1700
Cysteine
16
17
63
400
0.37
14
1600
383
228
19
22
870
644
> 1700
35
36
90
120
13
73
> 1900
37
2.9
78
24
430
43
> 1400
> 1200
50
51
300
101
110
9.5
> 1200
> 1100
> 1100
> 5100
Methionine
56
33
> 1300
Phenylalanine
59
31 f
230
130
14
87
170
350
350
0.6
244
2300
1200
228
> 1400
Glutamine
Isolewine
Leucine
38
39
40
43
Lysine
23
49
61
62
63
71
72
73
80
81
6-9
250-500
1040
1-3
6-9
3(cys+met)
> 1300
> 1700
> 1200
1500
17
19 29)
120-(ile+val)
620 (ile+leu)
12
200
500
> 1700
> 1400
> lo4
> 33 32)
370
500
K
> 14M)
> 1400
> 1400
> 1500
96 37)
continued on next page
the lowest I1 (< 12) and were classified as very potent antagonists (see Table 2). In most cases the structurally related
natural amino acid could reverse the antimetabolic effect.
But there were two exceptions: the effect of 63 - a supposed
phenylalanine antagonist - could be eliminated only by addition of a mixture of cysteine and another amino acid possessing a nucleophilic side chain like methionine, serine or
threonine. A possible explanation for this phenomenon is an
acceleration of the hydrolytic decomposition of 63 into two
acetic acid molecules and an inactive 2-nitro-l-phenyl-l,3propandiol fragment. This would be inactive since the corresponding 2-amino- 1-phenyl-1,3-propandiol (58) was without effect (MM) despite containing the more permissable 2amino- instead of the nitro- group.
Arch. Pharm. (Weinhcim) 322.847-852 (1989)
Similarly, the antiproliferative effect of 90 could not be
antagonized by the structurally related amino acids
phenylalanine or serine alone, but only by isoleucine in
combination with another branched amino acid (leucine or
valine). Both 63 and 90 showed no or only weak inhibitory
activity in the aminoacylation reaction of A A R S specific for
the putatively antagonized natural amino acids. These observations suggested an alternative mode of action for these
compounds.
Of the other three compounds most active in MHB 17.95,
and 96, only 95 had a strong inhibitory effect on A A R S . In
general, of all compounds tested only half of the strong
AARS inhibitors (Ki/Km < 2) could stop the proliferation of
E.coli. This was especially the case for the a-amino
850
h k e , Schonenberger, and Holler
Table 2 (continued)
Amino Acid
Antagonist
AARS
Ki/Kma
Proline
Serine
Threonine
Tryptophan
MIC (pM)
I1
M M ~
MHB'
M M ~
83
84
32 ~ 8 3 8 )
10 39)
20
1.8
> 5000
1200
> 2300
> loo0
90
91
0.6
73
670
> 1200
> 2100
3 41)
92
60
40
430
> 1900
> 1700
94
95
96
103
104
Tpsine
110
113
Valine
117
120
1.5 842)
1.3 g41)
9.7 842)
42)
48
42)
4.5
0.6
0.6
0.3
0.6
K
> 1200
576
576
> 2300
> 1200
29
120
150
500
58
3.3 P849
214 g4@
7.2
550
> 7400
> 2200
14-22
': Ratio of the inhibition constant of the antagonist (Ki) to the Michoelis-Menren-constant (Km) of the natural amino acid, determined in the
aminoacylation test unless otherwise stated
Minimal inhibitory concentration (MIC)in minimal medium (MM)
': Minimal inhibitory concentration (MIC)in Miiller-Hinton broth (MHB)
d : Inhibitory Index (11) determined in MM (see text)
e: 0.0028 P I 3 )
f : ICs (concentration required for 50% growth inhibition) = 15 pMI5)
J I8 C: !
<amino acid antagonist> / Km <amino acid>
h: ICH,= 82 pM")
i: ICx,= 77 pMIn
P : ATP-pyrophosphate exchange assay
x: acts as a weak substrate
y: neither inhibitor nor substrate
*: Ki calculated using data from literature according to the equation47): Ki = Km . (14) / i . (Km + S )
where I = concentration of the competitive inhibitor
i = degree of inhibition of the reaction rate
S = concentration of natural amino acid
Km = Michoelis-Menfen-constant of the natural amino acid
-: not determined
K: antagonism not detectable.
b:
alcohols 31, 42, 53, 78, and 115 (group 1-inhibitors;
Ki/Km 3.41°);1.6 'I); 0.4 12); 0.12 13) and 1.8 14), respectively),
which did not show any antibacterial effect. Most of the
A A R S inhibiting compounds, which were also growth inhibitors in h4M (MIC < 3pM) or MHB (MIC < lmM), were
a-amino carboxylic acids: 17, 37, 81, 84, 95, 96, and 110.
The only exception was the m i n e 59 being a very strong
competitive phenylalanyl-tRNA synthetase inhibitor (group
1, Ki/Krn = 0.01) and also a growth inhibitor (MIC = 31 pM
in MM and 244 pM in MHB).
Finally, two conclusions could be drawn: First, for compounds related i i their chemical structure to the natural
amino acids no simple correlation between inhibition of
AARS and of bacterial growth was evident. Second, the
compounds were only weak growth inhibitors in medium
containing competing natural amino acids (the best MIC
were in the range of 200 pM in MHB) and, therefore, were
unsuitable for an antibacterial chemotherapy (below 1 pM
would be desirable).
The authors thank E. Schreiber and H. Vilser for technical assistence.
Experimental Part
Melting points: BUchi 510 apparatus, uncorrected. 'H-NMR spectra:
Varian EM 360 A (60 MHz): TMS as internal reference.- Elemental analyses: Mikroanalytisches Laboratorium der Universitiit Regensburg; thc
results were within f 0.4% of the theoretical values. The purity of all corn
pounds was confirmed by tlc on Merck F254 silica plates.
Materials
Compounds 1,4, 28.41. 58,114, and 118 were obtained from Merck,
Darmstadt (FRG). 2, 6, 7. 10, 19, 21, 43, 81, 83, 98, 109, 117, 119, 120,
and 121 from Serva Heidelberg (FRG).13, 20, 34, 76. 88, 99, and 102
Arch. Pharm. (Weinheim) 322, 847-852 (1989)
85 1
Antiproliferative Effects of Amino Acid Antagonists
from Janssen Chimica, Nettetal (FRG), 74 from Riedel-deH%n, Seelze
(FRG),108 from EGA-Chemie, Steinheim (FRG),37, 39, and 111 from
Fluka, Neu-Ulm (FRG).
Compounds 9,12,15,20,40,44,45,48,65,85, and 86 were donated by
Dr. Engel, Degussa Phanna Gruppe, Frankfurt (FRG), 24 by the Burzynski
Research Institute, Houston, Texas, 30.64, and 66 by Dr.Holzinger, Bad
Abbach (FRG), 57 by Sir L. Fowden, Rothamsted Experimental Station,
Harpenden, Heas. (UK) and 63 by Egypt Pharmacochemical Works, Budapest.
49 was synthesized according to Tesser and Nef”ns16’17), 50 according
to D e Marco et al.’*); 59 was prepared as described by Kenvin et al.I9). 70
[N-(4-fluorobenzyl)-L-phenylalanine,yield 815,m.p. 216‘C; C1&I1,$NOz
(273.3); ‘H-NMR (TFA): qppm) = 3.55 (m; 2H, Ar-Cft-CH), 4.53 (m;
broad, 3H, Ar-Cft-NH- and a-CH), 7.05-7.67 (m;9H, ArH)] and 60 were
synthesized according to Quirt et aL2O), 61 and 62 as described by Anderson
and Santi”).
The following two compounds were prepared according to Meindl et
al?” (method A): 90 [N-(2,6-dichlorobenzylidene)ethanolamine,yield
71%; CgH9ClzN0(218.1); m.p. 38’C.- ‘H-NMR (CDCl3): 6 (ppm) = 1.72
(s; broad, -OH), 3.88 (s; 4H, -Cft-C&OH), 7.27 (“t”; 3H, Ar-H), 8.47 (s;
1H, -CH=N-)I and 68 ~-(2,6-dichlorobenzylidene)-2-phenylethylamine,
yield 86% m.p. 25’C; C15H13C12N (278.2).- ‘H-NMR (CDC13): 6 (pprn) =
3.05 (t; J = 7.1 Hz, 2H, -CHz-Ar), 3.96 (t; J = 7.1 Hz, 2H, =N-CHz-), 7.26
(s; 8H, Ar-H), 8.35 (s; IH, -CH=N-)I.
The following two compounds were prepared by reduction of the
described benzylidene derivates according to2*)(method C): 69 [N-(2,6-dichlorobenzyl)-2-phenylethylamineHC1, yield 685, m.p. 189’C;
C I ~ H & ~ (316.7),~N
‘H-NMR (cDc13): 6 (ppm) = 1.78 (s; lH, -NH-),
3.24 (S; 4H, -NH-C&-Cft-Ar), 4.50 (s; 2H, A r - a - N H - ) , 7.25 (s; 8H,
Ar-H)] and 89 [N-(2,6-dicNorobenzyl)ethanolamine,yield 60%. m.p. 55’C
(Lit. 57-59’?’)) CgHllClzNO (220.1).- ‘H-NMR (CDCI3): 6 (ppm) = 2.21
(S; 2H. NH- and -OH), 2.80 (ti J = 5.2 Hz, 2H, -NH-C&-CHzOH), 3.67 (ti
J = 5.2 Hz, 2H, -C&-OH), 4.12 (s; 2H, Ar-Cb-), 6.98-7.52 (m; 3H, ArH)].- 101 was prepared according to Preobrozhenskayh2). All other compounds were obtained by Sigma, Deisenhofen (FRG).
Biological Methods
a) Growth inhibition of E.coli B
The strain E.coli B was obtained from the Institut fur BD
tanik, Universitiit Regensburg (FRG). Minimal medium was
prepared according to Sunti et al. (basal salt medium)”),
Miiller-Hinton broth was obtained by Merck, Darmstadt
(FRG). Growth inhibition was measured as described23).
Antagonism was tested in minimal medium using antagonist
concentrations in the range of 1 up to 3 times the MIC. The
values of MIC and II listed in table 2 represent the mean of
2 - 4 separate experiments, which differed at the most by
one step in the geometric dilution test. 59 served as internal
standard in all experiments.
b) Preparation of amino acyl-tRNA synthetases”)
E.coli B in exponential growth phase was harvested,
washed with 0.9% NaCl solution and sonicated in standard
buffer containing 50 mM Tris . HC1 (pH 7.3), 5 mM MgClZ,
1 mM dithiothreitol, 1 m M phenylmethylsulfonyl fluoride
(PMSF), 0.1 mM EDTA and 10% (V/V) glycerin (at 4‘C,
also the following steps). Cell debris was removed by centrifugation at 5 OOO g, then 100 000 g. The supernatant was
treated with streptomycin 2’) and fractioned with ammonium
Arch. Pharm. (Weinheim) 322,847-852 (1989)
sulfate”). The protein fraction between 35% and 75% saturation of ammonium sulfate was dissolved in standard buffer containing 0.1 mM PMSF, dialyzed twice against a 1
000-fold volume of the same buffer and then concentrated
by dialysis against the buffer containing 50% (VN)
ethylene glycol. Preparations were stored at -20°C for not
longer than 6 months.
c) Assay of
Enzyme activity was determined by the aminoacylation
assay described by Kosakowski and Bock 26). Unfractionated
tRNA from E.coli was obtained from Boehringer, Mannheim (FRG). Inhibition constants listed in table 2 are mean
values of at least two separate experiments, which differed
by less than 10%.
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