3
PRELIMINARY NOTES 545 foie apr~s 6limination de l'acide trichlorac6tique par l'6ther k o ° ; la m6thode employ6e est celle d6crite par STREHLERET TOTTER 9, fond6e sur la luminescence de pr@arations d'organes lumineux de Photinus pyralis proportionnelle k I'ATP present. Les r6sultats obtenus, ont 6t6 r~sum6s darts le Tableau I. Chez le rat mMe, les injections de thyroxine provoquent une forte diminution du taux de I'ATP (de l'ordre de 7° %) sans modifier le poids du foie ; chez la femelle, la thyroidectomie entralne une forte diminution du poids du foie sans abaisser signifi- cativement le taux de I'ATP. De plus, on observe chez la femelle normale un taux de I'ATP plus dlevd que chez le mffie normal (3° % en plus; P ~ o.ooi); il n'est pas possible actuellement d'expliquer ce ph6nom~ne. Les r6sultats obtenus ~t propos de l'influenee des hormones thyroidiennes sont en accord avec une observation de VOGEL sur les 6rythrocytes de patients hyperthyroidiens, et avec les effets constat6s, sous l'influence de la thyroxine, dans le cerveau du rat par MILCU et al. 11 et dans le coeur par BERTOLINI et al. 12. Signalons cependant que CHILSON ET SACKS 13 n'ont pu mettre en 6vidence des modifications signifieatives du taux de I'ATP dans le foie du cobaye thyrotoxique. Les variations du taux de I'ATP selon l'~tat thyroidien de l'animal concordent avec les observations faites pr~c6demment sur le taux de PLP 8 et les niveaux d'activit6 des enzymes i~ PLP dans le foie 5-7. Ces r6sultats feront l'objet d'une publication ult6rieure plus d6taillde. Laboratoire de Chimie biologique, 96 Bld Raspail, Paris 6 ° FERNANDE CHATAGNER DANII~.LE GAUTHERON 1 p. HOLTZ, K. STOCK ET E. \VESTERMANN, Arch. exptl. Pathol. Pharmakol. Naunyn-Schmiedeberg's, 228 (i956) 322. 2 G. LITWACK, J. Biol. Chem., 228 (1957) 823. 3 A. HORVATH, Nature, 179 (1957) 968. 4 •. CANAL ET L. TESSARI, Boll. soc. ital. biol. sper., 33 (1957) 1472. 5 F. CHATAGNER, g. BERGERET ET J. LABOUESSE, Biochim. Biophys. Acta, 3° (1958) 422. t3. BERGERET, J. LABOUESSE ET F. CHATAGNER, Bull. soc. chim. biol., 4 ° (I958) 125. 7 13. JOLL/~S-BERGERET, J. LABOUESSE ET F. CHATAGNER, Bull. SOC. chim. biol., 42 (196o) 51. s j . LAnOUESSE, F. CHATAGNER ET ]~. JOLLI~S-BERGERET, Biochim. Biophys. Acla, 39 (196o) 372. 9 B. L. STREHLER ET J. R. TOTTER, dans D. CLICK, Methods o/ Biochemical Analysis, Vol. I, Interscience, N.Y., 1954, p. 341. 10 G. VOGEL, Klin. Wochschr., 36 (1958) 975; C.A., 53 (1958) I2456b. 11 ST M. MILCU, I. POTOP ETC. CIOClRDIA, Commun. Acad. rep. populare Romine; 7 (1957) 8t3; C.A. 52 (I957) i6551b. 12 A. M. BERTOLn~I ET F. QUARTO DI PALO, Boll. soc. ital. biol. sper., 32 (1956) 91. 13 O. P. CHILSON ET J. SACKS, Proc. Soc. Exptl. Biol. Med., lOl (1959) 331. Re~u le 13 mai 196o Biochim. Biophys. Acta, 41 (196o) 544-545 Discrepancy between thienylalanine activation and protein synthesis in bacteria Amino acid-activating enzymes are regarded as specific for those amino acids whose chemical structure and steric configuration render them capable of becoming building blocks of proteins. The correlation between amino acid activation and utilization for protein synthesis obtains even for certain unnatural amino acid analogs. It was Abbreviations: Tris, tris(hydroxymettlyl)aminomethane; RNA, ribonucleic acid. Biochim. Biophys. Acta, 41 (196o) 545-547

Discrepancy between thienylalanine activation and protein synthesis in bacteria

Embed Size (px)

Citation preview

Page 1: Discrepancy between thienylalanine activation and protein synthesis in bacteria

PRELIMINARY NOTES 545

foie apr~s 6limination de l'acide trichlorac6tique par l'6ther k o ° ; la m6thode employ6e est celle d6crite par STREHLER ET TOTTER 9, fond6e sur la luminescence de pr@arations d'organes lumineux de Photinus pyralis proportionnelle k I 'ATP present. Les r6sultats obtenus, ont 6t6 r~sum6s darts le Tableau I.

Chez le rat mMe, les injections de thyroxine provoquent une forte diminution du taux de I 'ATP (de l'ordre de 7 ° %) sans modifier le poids du foie ; chez la femelle, la thyroidectomie entralne une forte diminution du poids du foie sans abaisser signifi- cativement le taux de I'ATP. De plus, on observe chez la femelle normale un taux de I 'ATP plus dlevd que chez le mffie normal (3 ° % en plus; P ~ o.ooi); il n'est pas possible actuellement d'expliquer ce ph6nom~ne. Les r6sultats obtenus ~t propos de l'influenee des hormones thyroidiennes sont en accord avec une observation de VOGEL t° sur les 6rythrocytes de patients hyperthyroidiens, et avec les effets constat6s, sous l'influence de la thyroxine, dans le cerveau du rat par MILCU et al. 11 et dans le coeur p a r BERTOLINI et al. 12. Signalons cependant que CHILSON ET SACKS 13 n'ont pu mettre en 6vidence des modifications signifieatives du taux de I 'ATP dans le foie du cobaye thyrotoxique. Les variations du taux de I 'ATP selon l'~tat thyroidien de l'animal concordent avec les observations faites pr~c6demment sur le taux de PLP 8 et les niveaux d'activit6 des enzymes i~ PLP dans le foie 5-7.

Ces r6sultats feront l'objet d'une publication ult6rieure plus d6taillde.

Laboratoire de Chimie biologique, 96 Bld Raspail, Paris 6 ° FERNANDE CHATAGNER DANII~.LE GAUTHERON

1 p. HOLTZ, K. STOCK ET E. \VESTERMANN, Arch. exptl. Pathol. Pharmakol. Naunyn-Schmiedeberg's, 228 (i956) 322.

2 G. LITWACK, J. Biol. Chem., 228 (1957) 823. 3 A. HORVATH, Nature, 179 (1957) 968. 4 •. CANAL ET L. TESSARI, Boll. soc. ital. biol. sper., 33 (1957) 1472. 5 F. CHATAGNER, g . BERGERET ET J. LABOUESSE, Biochim. Biophys. Acta, 3 ° (1958) 422.

t3. BERGERET, J. LABOUESSE ET F. CHATAGNER, Bull. soc. chim. biol., 4 ° (I958) 125. 7 13. JOLL/~S-BERGERET, J. LABOUESSE ET F. CHATAGNER, Bull. SOC. chim. biol., 42 (196o) 51. s j . LAnOUESSE, F. CHATAGNER ET ]~. JOLLI~S-BERGERET, Biochim. Biophys. Acla, 39 (196o) 372. 9 B. L. STREHLER ET J. R. TOTTER, dans D. CLICK, Methods o/ Biochemical Analysis, Vol. I,

Interscience, N.Y., 1954, p. 341. 10 G. VOGEL, Klin. Wochschr., 36 (1958) 975; C.A., 53 (1958) I2456b. 11 ST M. MILCU, I. POTOP ETC. CIOClRDIA, Commun. Acad. rep. populare Romine; 7 (1957) 8t3;

C.A. 52 (I957) i6551b. 12 A. M. BERTOLn~I ET F. QUARTO DI PALO, Boll. soc. ital. biol. sper., 32 (1956) 91. 13 O. P. CHILSON ET J. SACKS, Proc. Soc. Exptl. Biol. Med., lOl (1959) 331.

Re~u le 13 mai 196o Biochim. Biophys. Acta, 41 (196o) 544-545

Discrepancy between thienylalanine activation and protein synthesis in bacteria

Amino acid-activating enzymes are regarded as specific for those amino acids whose chemical structure and steric configuration render them capable of becoming building blocks of proteins. The correlation between amino acid activation and utilization for protein synthesis obtains even for certain unnatural amino acid analogs. It was

Abbreviat ions: Tris, t r i s (hydroxymet t ly l )aminomethane ; RNA, ribonucleic acid.

Biochim. Biophys. Acta, 41 (196o) 545-547

Page 2: Discrepancy between thienylalanine activation and protein synthesis in bacteria

546 PRELIMINARY NOTES

pointed out by SHARON AND LIPMANN 1, for example, that certain t ryptophan analogs which were activated by a mammalian enzyme 1 were also utilized for net protein synthesis in bacteria 2. Such a correlation should be demonstrable with stronger reason when analog activation and utilization for net protein synthesis are parameters of one and the same organism.

We have found recently, however, that fl-2-thienylalanine was strongly activated by an enzyme extract from the phenylalanineless strain C-2 of Escherichia coli but unexpectedly was not utilized for appreciable net protein synthesis by this organism.

A bacterial enzyme extract was prepared by grinding 25-50 g of washed wet E. coli C-2 with alumina, extraction with 0.05 M Tris buffer (pH 7.2), differential centrifugation, exhaustive dialysis of the 92,00o × g supernatant, followed by lyophilization and reconstitution of the solid in a small volume of Tris buffer. Amino acid activation by this enzyme extract was assayed by measuring hydroxamic acid formation after the method of DAVlE el al. 3 Phenylalanine hydroxamic acid to be used as a colorimetric standard was prepared by a modification of the procedure of CUNNINGHAM el al. 4. Under the conditions of the assay I.O Fmole of phenylalanine hydroxamic acid yielded an absorbancy of 0.20, read in a Io mm cuvette of a Beckman Model DU spectrophotometer at 500 inf .

The bacterial-enzyme extract activated both phenylalanine and thienylalanine as depicted in Fig. i. The activation was ATP-dependent; control experiments with heat-inactivated enzyme preparations produced no formation of hydroxamic acid. Activations of thienylalanine and selected amino acids were additive. This is illustrated with methionine by data listed in Table I. In contrast, activation of thienylalanine and phenylalanine was non-additive (Table I), suggesting that both amino acids competed for the same activating enzyme.

The intact bacteria did not form appreciable quantities of protein when thienyl- alanine was substituted for phenylalanine ('fable II). No RNA was synthesized by thienylalanine-supplied E. coli C-2.

,090 [

.075

.060

.045

.030

.015

OD 5 0 0

x

x x

o

15 30 45 60 75

TIME IN MINUTES

J 90

Fig. i. Fiydroxalnate format ion catalyzed by an enzyme extrac t from E. coli C-2. The assay procedure was t ha t of DAVIE et al. s. The diagram shows the typical rat io between the reaction rates for phenylalanine and thienylalanine hydroxamic acid formation as observed in numerous experiments . Linear i ty of the reaction with t ime was established separately. The enzyme extract

exhibited s t rong pyrophospha tase activity.

Biochim. Biophys. Acta, 4 ~ (I96o) 545-547

Page 3: Discrepancy between thienylalanine activation and protein synthesis in bacteria

PRELIMINARY NOTES 547

T A B L E I

A D D I T I V I T Y OF T H I E N Y L A L A N I N E A N D M E T H I O N I N E A C T I V A T I O N S A N D N O N - A D D I T I V I T Y OF

T H I E N Y L A L A N I N E A N D P H E N Y L A L A N I N E A C T I V A T I O N

Exper imen ta l mix tures contained i mmole NH2OH, 3 ° /~moles Tris, p i t 7.2, io /~moles MgC1 v and IO #moles ATP in addit ion to the amino acids listed below in a to ta l volume of i . i ml. In- cubat ion was carried out for 6o min at 37 °. DL-thienylalanine concentrat ions were twice those of na tura l L-amino acids in allowance for the racemic composi t ion of the analog preparat ion.

Concentration Hydroxamate formed Compound activated (raM) (absorbancy at 500 ml~)

DL-Thienylalanine 17.4 o. 12 i L-Methionine 8.7 o.i 63 L-Methionine 17.4 o. 185 oL-Thienylalanine + 17.4

L-Methionine 8.7 o.284

DL-Thienylalanine 14.6 o.o95 L-Phenylalanine 7.3 o. 12 i L-Phenylalanine 14.6 o. 155 nL-Thienylalanine + 14.6

L-Phenylalanine 7.3 o.i 34

T A B L E I I

P R O T E I N S Y N T H E S I S IN E. coli C-2

Exponent ia l ly growing cultures were washed exhaust ively and resuspended in fresh glucose- mineral medium with the additions indicated below. Samples were taken at t ime intervals, the collected bacter ia were extracted wi th hot trichloroacetic acid, and the residues were solubilized in o.I N N a O H and analyzed for nitrogen by H2SO~-HaO e digestion followed by Nesslerization.

Prolein (per cent of initial values) Time (rain) no phenyla2a~tine 5 ° I~g[ml zo pg/ml

nor analog DL-thienyiaianine L-phenylalanine

O IOO IOO IOO

60 83 lO 7 196 12o IOO lO 7 346 18o IOO lO 7 - -

The inability of thienylalanine to support significant protein and RNA net biosynthesis contrasts markedly with the rate and extent of activation of this analog by the bacterial enzyme extract. This discrepancy constitutes the essential subject of the present communication, and will also be the subject of future communications.

Department of Molecular Biology, Walter Reed Army Institute of Research,

Washingto~ 12, D.C. (U.S.A.)

ALAN DAVID WOLFE

FRED E . HAHN

Received April Ilth, 196o

1 l~. SHARON AND F. LIPMANN, Arch. Biochem. Biophys. , 69 (1957) 219. 2 A. B. PARDEE, V. G. SHORE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 21 (1956) 406. a E. W. DAVIE, V. V. KONINGSBERGER AND F. LIPMANN, Arch. Biochem. Biophys. , 65 (1956) 21. 4 K. G. CUNNINGHAM, G. T. NEWBOLD, F. S. SPRING AND J. STARK, J. Chem. Soc. (London),

(1949) 2o91.

Biochim. Biophys. Acta, 41 (196o) 545-547