Documente Academic
Documente Profesional
Documente Cultură
68, 1 - 3 (1976)
PI.
Action of Indolmycin
-t/---I;
50
' 0
20
<
40 60 80 100
KCI concentration [ m M I
100
200
300
4M)
500
per assay [ k g ]
tRNA
5 mM
2 ' 0
P
=l-
2
4
6
8
ATP concentration [rnM]
10
5
10
15
20
5150 enzymes [ E . c o l i ] per assay [PI]
10
20
30 40 50
Incubation time [min]
60
using tRNA from calf liver and 300000 x g supernatant from rat liver (see Fig. 2B). 10% inhibition is
seen at an indolmycin concentration of 30 pM, and
only 30% at 10 mM. The possible breakdown of
indolmycin by enzymes present in the crude rat liver
extract was tested by the following experiment.
Indolmycin was incubated in the presence of rat liver
extract at 30 "C for 60 min and the indolmycin activity
was examined before and after incubation. Similarly
indolmycin was incubated alone at 30 "C for 60 min
and the activity tested before and after incubation.
Activity was determined in vivo by measuring the
growth inhibition of Staphylococcus uureus. Incubation of the drug with and without rat liver extract did
not affect the indolmycin activity indicating that
indolmycin is not inactivated during incubation in
presence of rat liver extract. A hybrid system composed
of calf liver tRNA and 150 000 x g supernatant from
E. coli is inhibited even more strongly than the prokaryotic system (Fig. 2C). Thus, the inhibition of
indolmycin is related to the synthetase and not to the
tRNA fraction. Most likely, indolmycin competes
with tryptophan on the respective synthetase.
The last .experiment was designed to analyze the
type of inhibition (see [S]). The tryptophanyl-tRNA
In
1I [Tryptophan] (nM-' )
50t 1
4
0.001
0.01
0.1
10
Burk diagram the inhibition constant (Ki) of indolmycin was calculated, for both concentrations to have
a value for K , = 8 - 9 x
M.
In conclusion, indolmycin specifically inhibits
prokaryotic tryptophanyl-tRNA ligase whereas the
corresponding eukaryotic ligase is only slightly affected. The effect of indolmycin on mitochondria (penetration into mitochondria, inhibition of the mitochondrial aminoacylation) was not analysed. It may
turn out that the drug can be used in vivo as a bacteriospecific agent, even if it inhibits mitochondria1 aminoacylation in vitro, if it does not penetrate into the mitochondria.
We thank Drs H . G. Wittmann and R. Brimacombe for advice
and helpful discussion.
REFERENCES
1 . Vazques, D. (1 974) FEBS Lett. 40 S. 63 84.
2. Nass, G., Poralla, K. & ZHhner, H. (1969) Biochem. Biophqx.
Rc~s.Commun. 34, 83-91.
3. Schach von Wittenau, M. & Els, H. (1963) J . A m . Cheni. Soc.
85, 3425 - 3431,
4. Preobrazhenskaya, M. N., Uvarova, N. V., Padeiskaya, E. N.,
Pershin, G. N. & Suvorov, N. N. (1967) Dokl. Alrud. Nuuk.
SSSR, 172, 870-872.
5. Preobrazhenskaya, M. N.. Balashova, E. G., Turchin, K. F.,
Padeiskaya, E. N., Uvarova, N. V., Pershin, G. N. & Suvorov, N. N. (1968) Tetrahedron, 24, 6131 -6143.
6. Chan, T. H. &Hill, R. K . (1970) J . Org. Chem. 35, 3519-3521.
7. Ulbrich, B. & Nicrhaus, K . H. (1975) Eur. ./. Bioc~hem.57,
49 54.
8. Dixon, M. 6i Webb, E. C. (1964) Enzymes, 2nd edn, pp. 315359, Longmans Green and Co., London.
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R. G. Werner*, L. F. Thorpe, and W . Reuter, Dr. Karl Thomae GmbH, D-7950 Biberach an der Riss,
Federal Republic of Germany