- Email: [email protected]
Incorporation of p-fluorophenylalanine into proteins of Lactobacillus arabinosus
3I~
BIOCHIMICA ET BIOPHYSICA ACTA
INCORPORATION OF p-FLUOROPHENYLALANINE OF LACTOBACILLUS
VOL. 2 8 ( I 9 5 ~ )
INTO PROTEINS
ARABINOSUS*
R. S. B A K E R * * , J O S E P H E. J O H N S O N * * * AND S I D N E Y XV. FOX§
Chemical Laboratory, Iowa State College, Ames, Iowa (U.S.,4,)
The concept that the phenotypic expression of an organism may be correlated with the identity of its constituent proteins, has been in the literature for decades 1. Any alteration in the content of any one amino acid in a protein molecule might accordingly be related to a detectable alteration in the organism. A number of attempts to alter constitution of total proteins by control of the diet or medium have been recorded ~-s. Some of the claims of successful alteration of this kind have been refuted 1°-~4. The reported findings fail to meet the stated experimental objective for one or two main reasons: (I) only inadequately precise methods of amino acid assay were available for use and (2) a change in amino acid composition of an entire organism or tissue may be explained simply as a shift in proportion of proteins rather than as an alteration in composition of one or more proteins. Accordingly, the use of amino acids containing groups not found naturally becomes of interest. To the extent that it might be established that such an unnatural amino acid is incorporated into protein ~5, compelling evidence of the alterability of protein synthesis would be at hand. A substance which early received similar study is found in the work of DYER~6. DYER synthesized the now highly useful methionine antagonist, ethionine, and fed it to rats. She discovered thereby an early antimetabolite. Evidence for incorporation of ethionine into protein has been presented ss. Parenthetically, it may be noted that the sulfur of ethionine is metabolically available ~v. The suggestion of incorporation of selenocystine into protein has been offered ~8. Selenocystine may however be looked upon as a natural amino acid. The examples given indeed indicate that there is not an a priori qualitative distinction between natural amino acids and unnatural amino acids. The deciding factors may be merely the identities of the amino acids in the environment and the opportunity for the organism to adjust to them. Results most easily interpreted as incorporation of an unnatural or unusual amino acid were reported with p-fluorophenylalanine (FPA) in 195 I15. This appears to * J o u r n a l P a p e r No. J-3284 of the I o w a Agricultural and H o m e Economics E x p e r i m e n t Station, Ames, Iowa. Project t [ Tt, s u p p o r t e d in p a r t by the Rockefeller Foundation. F r o m the M.S. thesis (i951) and the P h . D . thesis (1953) of R. S. BAKER. ** Present address: Eli Lilly and Co., Indianapolis, Ind. * * * Present address: Chemistry 1)epartment, Massachusetts I n s t i t u t e of Technology, Cambridge, Mass. § Present address: C h e m i s t r y l ) e p a r t m e n t and Oceanographic Institute, l'lorida State University, Tallahassee, Fla.
]{e[erences p, 327.
VOL. 2 8
(1958)
p-FLUOROPHENYLALANINE IN PROTEINS
319
be the first recorded instance of the incorporation of an unnatural amino acid into the protein of an organism ~9. An earlier, doubtful possibility of the incorporation of D-alanine ~° has been shown to involve an amino acid which is definitely natural in its n form2h More conclusive evidence of incorporation of FPA into proteins of L. arabinosus was presented in 195 4 by way of partial hydrolysis followed by blocking N-terminal and free amino acids with phenylisothiocyanate and total hydrolysis of the peptides remaining 22. These experiments and subsequent experiments are described in detail in this paper. MUNIER AND COHN23 have recently reported the incorporation of p-fluorophenylalanine, and of thienylalanine, into the proteins of Escherichia coll.
Besides the unlikelihood of its natural occurrence, p-fluorophenylalanine was selected for the present study because of the small halogen atom and the consequent similarity of FPA to the aromatic amino acids phenylalanine and tyrosine, because of the unlikelihood of transfer of a w l fluoride to another amino acid 24, and because of the presumed feasibility of selectively following fluorine by elemental analysis, in contrast to determining altered ratios of natural amino acids in proteins. In practice the high RF of F P A in the solvents employed proved to be more useful in tracing it; in a developing solvent of tert.-butyl a l c o h o l - m e t h y l ethyl k e t o n e - w a t e r (4: 4: 1.5), FPA ran ahead of all amino acids in protein hydrolyzates.
METHODS
Organisms Laetobacillus arabinosus (ATCC 8o14) w a s m a i n t a i n e d as p r e v i o u s l y de s c ri be d, as w e re p h e + t y r +, t y r +, a n d t h e F P A - t o l e r a n t s t r a i n 15. L. arabinosus \ V r i g h t w a s a bl e t o s y n t h e s i z e its t r y p t o p h a n ; it w a s o b t a i n e d f r o m Dr. L. D. WRIGHT 25. I t di d n o t lose i t s a b i l i t y t o s y n t h e s i z e tryptophan when the stock culture was transferred.
Culture o[ organisms The b a s a l m e d i u m of KUIKEN et al. ze w a s used w i t h t h e p r o p e r a m o u n t s of P A a n d F P A a d d e d . F o r p l a t e a s s a y s , w a s h e d a g a r w a s a d d e d (I3/4 %). The m e d i u m w a s a n t o c l a v e d for 15-2o m i n a t 15 lb. p r e s s u r e a n d i n c u b a t e d a t 3 6 - 3 7 °. G r o w t h w a s m e a s u r e d in IO ml c u l t u r e s in o p t i c a l l y m a t c h e d P y r e x t u b e s ( i i x 15 ° ram) w i t h a Model I i C o l e m a n U n i v e r s a l s p e c t r o p h o t o m e t e r , a t 575 mtt.
Preparation o[ cy/olyzate \Vhen g r o w t h h a d ceased, t h e cells were collected b y c e n t r i f u g a t i o n iu a S h a r p l e s c o n t i n u o u s c e n t r i f u g e . The cells were s u s p e n d e d in p h y s i o l o g i c a l s a l i n e o v e r n i g h t . A f t e r c e n t r i f u g i n g , t h e cells were r e s u s p e n d e d in saline a n d c e n t r i f u g e d agai n. The cells w e r e c y t o l y z e d b y g r i n d i n g w i t h s a n d a n d e t h e r ; t h e e t h e r w a s d e c a n t e d a n d e x t r a c t e d s e v e r a l t i m e s w i t h w a t e r . The a q u e o u s e x t r a c t s were c o m b i n e d w i t h t h e c y t o l y z a t e .
Blocking o[ [ree amino acids N i t r o g e n w a s d e t e r m i n e d in t h e c y t o l y z a t e b y m i c r o - K j e l d a h l , a n d p h e n y l i s o t h i o c y a u a t o (PTC) w a s a d d e d 27 on t h e b a s i s t h a t o n e - h a l f of t h e n i t r o g e n w a s p r e s e n t as flee a m i n o g r o u p s w i t h an a d d i t i o n a l n i n e t e e n - f o l d excess. An e q u a l v o l u m e of p y r i d i n e w a s a d d e d t o an a l i q u o t of t h e c y t o l y z a t e a n d t h e m i x t u r e w a s m a d e a l k a l i n e to b r o m o t h v m o l bl ue w i t h N a O H . The PTC w a s a d d e d a n d t h e r e a c t i o n k e p t a l k a l i n e b y t h e p e r i o d i c a d d i t i o n of N a O H w h i l e t h e r e a c t i o n w a s i n c u b a t e d a t 37 °. \Vhen t h e c o n s u m p t i o n of a l k a l i ceased, t h e excess p y r i d i n e a u d PTC w e r e r e m o v e d b y e x t r a c t i o n w i t h five v o l u m e s of benzene. The r e m a i n i n g m i x t u r e w a s h y d r o l y z e d w i t h an e q u a l v o l u m e of cone. HCl.
Paper chromatography P a p e r c h r o m a t o g r a m s were rim on \ V h a t m a n No. t filter p a p e r in s h e e t s of 7 i~ "~ q 14 i nc he s in c h a m l ) e r s which were .5-11). c h e m i c a l - r e a g e n t b o t t l e s w i t h s c r e w caps. A c y l i n d e r w a s m a d e of t h e p a p e r b y s t a p l i n g t h e t w o o p p o s i t e edges t o g e t h e r w i t h o u t t h e e dge s t o u c h i n g . F or o p t i u l u n l
Re#'rences p. 327.
320
R.S.
BAKER,
J. E. JOHNSON,
S. W . F O X
VOL. 28
(I958)
s e p a r a t i o n of F P A a n d P A t h e s o l v e n t of t e r l . - b u t y l a l c o h o l - m e t h y l e t h y l k e t o n e - w a t e r ( 4 : 4 : 1 . 5 b y v o l u m e ) w a s r u n to t h e t o p of t h e p a p e r 3 t i m e s w i t h t h e c y l i n d e r r e m o v e d f r o m t h e c h a m b e r a n d dried b e t w e e n each a s c e n t to t h e top. F or q u a n t i t a t i v e r e s u l t s t h e p a p e r c h r o m a t o g r a m w a s d i p p e d i n t o o.2 % n i n h y d r i n in a c e t o n e a n d a l l o w e d t o d r y in a i r a t room t e m p e r a t u r e . E s t i m a t i o n w a s m a d e b y v i s u a l c o m p a r i s o n of color i n t e n s i t i e s of u n k n o w n s a m p l e s with standards. Inhibition assay
T h e zone of i n h i b i t i o n c a u s e d b y F P A on L. a r a b i n o s u s p h e + t y r + w a s c o m p a r e d w i t h t h e a r e a of i n h i b i t i o n of t h e u n k n o w n s a m p l e s . The F P A w a s a d d e d t o t h e a g a r p l a t e b y p l a c i n g t h e filter p a p e r d i s k c u t f r o m a c h r o m a t o g r a m o n t o t h e p r e v i o u s l y i n o c u l a t e d a n d solidified m e d i u m . P h e n y l a l a n i n e a n d t y r o s i n e were o m i t t e d from t h e m e d i u m for t h i s o r g a n i s m . The p a p e r d i s k c o n t a i n i n g t h e F P A w a s c u t on t h e b a s i s of t h e l o c a t i o n of F P A on a c o m p a n i o n c h r o m a t o g r a m w h i c h h a d been t r e a t e d w i t h n i n h y d r i n . The a r e a of i n h i b i t i o n w a s r e l a t e d to t h e a m o u n t of L - F P A *. RESULTS
The RF values of phenylalanine, p-chlorophenylalanine, and FPA are given in Table I. It is seen that each of the halophenylalanines travels ahead of phenylalanine, which is the fastest of the amino acids from protein hydrolyzates, in this solvent mixture. Of the two unnatural amino acids, the chloro analog exhibits the higher RF. In Fig. • are presented the results of a standard assay of FPA by inhibition analysis. A fresh standard was prepared for each estimation. In each case, the plot tended to be linear in the range in which values had meaning in the experiments. TABLE
I
~ F VALUES OF P H E N Y L A L A N I N E , p - C H L O R O P H E N Y L A L A N I N E , AND p - F L U O R O P H E N Y L A L A N I N E IN /e~'/.-BUTYL ALCOHOL METHYL ETHYL K E T O N E - - W A T E R ( 4 : 4 : i . 5 ) A m i n o acid
RF
Phenylalanine p-Chlorophenylalanine p-Fluorophenylalanine
i
15-
~
0.60 0.84 o. 76
,
•
,z O
ilJ o o 0 ]" 1100
I I ; 1200 1300 1400 AREA OF INHIBITION IN SQUARE M I L L I M E T E R S
1500
Fig. r. P l o t of a r e a of i n h i b i t i o n in d i s k a s s a y vs. e o n c e n t r a t i c m of ~r_-/~-fluorophenylalanine. * O t h e r e x p e r i m e n t s w i t h k n o w n a m o m l t s of free F P A m i x e d w i t h a p o r t i o n of t h e c y t o l y z a t e of o r g a n i s u l s g r o w n in t h e a b s e n c e of F I ' A s h o w e d t h a t 85-90 % of t h e free F P A could be b l o c k e d by this method. l¢e/ereJ*ces p. 327 .
VOL. 28 (1958)
p-FLUOROPHENYL\L\NINE
321
IN PROTEINS
60
S
2/
70-
¢ 80-
90-
/ 100 -
~__~z 10
/
I I ! I I I I ! I ! t
I
n I
O"F'
20
30
40
INCUBATION
TIME
J 5O
60
"to
80
(HOURS)
Fig. 2. L. arabinosus g r o w n in m e d i u m c o n t a i n i n g a i o : i ratio of p - f l u o r o p h e n y l a l a n i n e to p h e n y l alanine. T y p i c a l t i m e - g r o w t h r e l a t i o n s h i p of L. arabinosus on c u l t u r i n g (solid curve) a n d s u b c u l t u r i n g ( d o t t e d curve) in t h e presence of p - f l u o r o p h e n y l a l a n i n e , o.o8 t, moles p h e n y l a l a n i n e per t u b e , o.8 t, moles p - f l u o r o p h e n y l a l a n i n e p e r tube.
In Fig. 2 are presented the results of subculturing parental strain in the presence of an inhibitory proportion of F P A (IO F P A : I PA on a DL molar basis). It is clear t h a t the parental strain grows only after an extended lag period. Once the surviving adjusted cells are reinoculated into medium containing F P A at the parentally inhibitory level, initiation of growth is as rapid as in the controls. This evidence substantiates the conclusion, based on the phenomenon of haphazard appearance of F P A tolerance 15, that the tolerant strain is a naturally selected m u t a n t . These results are for FPA-tolerance. The phenomenon of stimulation b y subinhibitory ratios of F P A has exhibited no time lag in a n y of the numerous trials. No evidence is at h a n d t h a t stimulation of the parental strain involves mutation, nor is there any evidence for the puzzling interpretation b y others t h a t the organism develops a requirement for the analog in these experiments 2s. On the contrary, the early evidence was interpretable as direct utilization of F P A b y the parental strain 15. "Fable I I reveals trends which have been observed also in a n u m b e r of other experiments. The FPA-tolerant strain is virtually unaffected b y growth in subinhibitory concentrations of FPA. The parental strain however demonstrates stiinulation. The difference between these two was larger in other trials. The other three strains of L. arabinosus suffered marked inhibition at this level. Tile sensitivity to inhibition b y F P A b y three of the strains also explains w h y one of these, t)he + tyr+, was selected for use in inhibition assays. Table I I I represents typical stimulation due to F P A at limiting concentrations of PA. This type of result was obtained repeatedly. Tables I I I and IV present the results of large scale stimulation experiments. 111 a typical one of these, 3 ° 1 of medium containing 39.6 mg I)L-PA (phenylalaifine) and 98.6 mg 1)L-FPA, was harvested after 48 h of incubation of L. arahim~szls at 3T'The cells were cytolyzed; to the final vohituc of 3oo ml an equal voluine ol pyridine aud 5 ° g of PTC was added. The reaction was considered to be complete when consumption of alkali had stopped; the solvent was then removed by distillation under reduced pressure. The residue was resuspended in 1oo ml of distilled water which Re/erences p. ,72 7.
322
R.S.
B A K E R , J . E. J O H N S O N , S. W . I;OX TABLE
VOL.
FPA/PA molar ratio
(1058)
II
GROWTH RESPONSE OF VARIOUS STRAINS OF L a c l o b a c i l l u s TO p-FLUOROPHENYLALANINE *
Strain
28
arabinosus
Incubation time (hours) 7
x6
24
37
4°
48
55
Parental
o 2.5
91 92
62 69
59 53
56 49
-44
-44
---
Tyr +
o 2.5
93 --
57 --
47 95
4° 94
34 83
31 71
-65
Phe + tyr +
o 2.5
92 --
42 --
21 91
18 88
16 72
--61
-54
Wright
o 2.5
92 93
8o 86
63 81
61 80
-81
-80
-77
FPA-Tolerant
o 2-5
98 97
86 83
66 63
60 58
60 56
---
---
PA =
phenylalanine,
FPA
-- fluorophenylalanine
* V a l u e s a r e g i v e n in p e r c e n t t r a n s m i s s i o n , a n d a r e a v e r a g e s o f q u a d r u p l i c a t e D L - P A p r e s e n t is a t l e v e l o f o . o 8 / * m o l e p e r t u b e . TABLE GROWTH RESPONSE OF L . a r a b i n o s u s
UNDER LIMITING CONDITIONS
Incubation time in hours
nL-FPA/tube*
24
31
48
o 0.20
56 5°
54 46
53 45
PA = * Each tube contains duplicate io-ml cultures.
III
TO F P A
Micromoles o/
phenylalanine,
0.08/,mole
io-ml cultures
FPA
=
fluorophenylalanine
of D L - P A . V a l u e s a r e p e r c e n t t r a n s m i s s i o n ;
TABLE
averages
of
IV
BIOLOGICAL ASSAY FOR TOTAL AND AMINO-BOUND F P A IN ALIQUOTS OF L . a r a b i n o s u s CYTOLYZATE SUBJECTED TO PARTIAL HYDROLYSIS, P r C TREATMENT, AND TOTAL HYDROLYSIS Culturist
Assayist
Total FPAI20 I~l
RSB RSB MM
RSB JEJ JEJ
IO /*g* 15 (16.2, 15.5, 14.5) 15 ( 1 5 . i , 15.6 )
Amino-bound FPA/20 Id 8 /*g* i o (lO.6, ~o.6, 2o.6, 9.8) i i (12.o, l I . 2 , IO.6)
* By paper-chromatographic methods, i2/*g FPA/2o/*1 were found for the total FPA and 6 / * g F P A / 2 o / * 1 f o r t h e a m i n o - b o u n d F P A . F P A -- p - f l u o r o p h e n y l a l a n i n e , PTC = phenylisothiocyanate.
was then r e m o v e d u n d e r r e d u c e d pressure. T h e residue was h y d r o l y z e d b y s u s p e n d i n g in 2oo ml of distilled w a t e r a n d 2oo ml of conc. HC1 a n d refluxing for 24 h. A f t e r t h e acid was r e m o v e d u n d e r r e d u c e d pressure, the residue was again s u s p e n d e d in distilled w a t e r a n d t a k e n to dryness again. The residue was e x t r a c t e d with 25o ml of d i e t h y l ether a n d 2oo ml of distilled water. The e t h e r l a y e r was s e p a r a t e d a n d w a s h e d with two 5o-ml p o r t i o n s of distilled water. T h e e t h e r washings were a d d e d ReJerences p. 327,
VOL. 2 8 (I(~58)
p-FLUOR()PHENYLALANINE IN PROTEINS
323
aho
O
QO
[bO dZT-'o
Fig. 3. Chromatographic analysis of hydrolyzate of L. a r a b i n o s u s . Left to right. Phenylalanine; Phenylalanine and p-fluorophenylalanine; Sample F, (see text); Sample F and p-fluorophenylalanine; pfluorophenylalanine; Sample I (see text); Sample I and added p-fluorophenylalanine. The dotted lines, the solid lines, and the filled in areas represent the increasing intensity of ninhydrin reacting areas.
O
c=3 PA
PA FPA
F
o F and FPA
FPA
I
O I and FPA
to the residue remaining in the aqueous layer of the extraction procedure. Two and one-half g of activated Norite A ~9was added to the aqueous solution and the mixture was shaken mechanically for 4 h. The filtered charcoal was eluted by stirring with 250 ml of 20 % aqueous acid containing 5 % phenol. After the charcoal was filtered, the filtrate was extracted with 300 ml of diethyl ether to remove the excess phenol and the ether extract was washed with ioo ml of distilled water which was combined with the filtrate and taken to dryness under reduced pressure. The residue was suspended in 50 ml of distilled water, centrifuged, the supernatant was poured off, and the residue was resuspended in distilled water and centrifuged again. The supernatants were combined and concentrated under reduced pressure. The precipitate which formed was centrifuged, washed with a small quantity of hot distilled water, the supernatant was combined with the concentrated solution and the pH was adjusted to 6 with 0.2 N NaOH. On the chromatograms in Figs. 3 and 4 this is sample F. Two controls were employed to determine the efficiency of the PTC treatment for the removal of free FPA .and to make certain the FPA could be detected if present in the protein hydrolyzate. For these controls, L. arabinosus cells grown on a complete synthetic medium without FPA were used and the procedure was the same as used in the previous experiment. In control G, 8 mg of DL-FPA was added to the cytolyzate before the treatment with PTC in order to see if the treatment blocked the free amino acids. In control sample H, 8 mg of DL-FPA was added after PTC treatment in order to ascertain if FPA, which was known to be present, could be detected. Control I did not contain any FPA. The results are shown in the chromatograms in Figs. 3 and 4. Ill Fig. 3, the chromatogram shows the following: (i) PA and FPA are readily separated, (ii) sample F contains a ninhydrin spot which has the same RF as FPA and is intensified R e / e r e n c e s p. 327 .
3°-4
R.s.
Ik\I,iER, J. E. JOHNSON, S. W. FOX
Q e
VOL. 2 8 (I05(~)
o
0 o
©o
0
£.
~
.;
,2-2,
i t
t
' ,
'i i:Z25 ! : Q)
i i
6
c~
0
xCJ PA
G
H
FPA
F
I
FPA
Fig. 4. C h r o m a t o g r a p h i c s e p a r a t i o n of h y d r o l y z a t e s of s a m p I e s F, G, H, a n d I. L e f t to right. P h e n y l a l a n i n e ; S a m p l e G (see text) ; S a m p l e H (see text) ; p - P l u o r o p h e n y l a l a n i n e ; S a m p l e F (see t e x t ) ; S a m p l e I (see t e x t ) ; p - F l u o r o p h e n y l a l a nine. T h e d o t t e d lines, t h e solid lines a n d t h e enclosed a r e a s r e p r e s e n t a r e a s of increasing intensities of n i n h y d r i n reaction.
PA
when F P A is added to sample F. A similar ninhydrin spot does not result in control I unless F P A is added to it. In Fig. 4, the c h r o m a t o g r a m shows the following : (i) control sample G does not show a ninhydrin spot for F P A indicating that PTC treatment removed the free F P A added to the extent that it can be determined b y qualitative paper-chromatographic methods, (ii) control sample H shows the presence of FPA, indicating that the acid hydrolysis and subsequent procedures do not cause an appreciable loss of,FPA, (iii) sample F shows the presence of F P A and control sample I does not show F P A . In order to assess the quantitative extent of the blocking of free F P A b y PTC 2.2i m g o t I)I,-FPA were treated with PTC and hydrolyzed according to the procedures outlined above. After removal of the acid, the residue was suspended in distilled water and taken to dryness. The residue was suspended in o.3 ml of distilled water and brought to p H 5 with o.4 ml of o.o5 N N a O H and assayed. After chromatographing the material with standard quantities of FPA, it was estimated that lO% of the F P A was not blocked b y PTC treatment. A second experiment was set up like control sample (i except that 30 mg of I)L-FPA was added to a cytolyzate preparation from L. arabinosus grown in a complete medium without FPA. This was treated with PTC, hydrolyzed, and extracted with ether as before and the mixture diluted to 5 ° ml and used for assay. Charcoal absorption and elution was omitted. Chromatograms failed to resolve the amino acids sufficiently for a quantitative comparison of ninhydrin colors with F P A standards; accordingly, bioassay was used. This assay indicated that I0°{, of the free F P A was not blocked by PTC treatment. IDI~NTIFIC.VI'ION OF AMINO-I~OUNI~ F P A IN PEPTIDES FROM L. arali.noslts
The experiments on the blocking of free F P A indicate that the blocking was 8 4 - 9 o % Re/erences p. 327.
VOL. 2 ~ ( I 9 5 8 )
p-FLUOROPHENYL.KLANINE IN PROTEINS
325
complete. To determine if the amount of F P A found in the cytolyzate of ceils grown under stimulating conditions was more or less than could be attributed to incomplete blocking of free FPA, quantitative estimations of the total F P A and the amino-bound F P A were made on peptides obtained by the partial hydrolysis of the cytolyzate. The partial hydrolysis presumably also served to liberate free amino acids trapped in proteins, but not in peptide linkage. After incubation in 3o 1 of medium containing 39.6 mg DL-PA, and 98.9 mg DI~-FPA for 48 h the L. arabinosus cells were harvested. Growth was followed in Io-ml cultures containing the same medium and the same medium without FPA. The cells were cytolyzed as described above and to the 25o-ml volume, 250 ml of conc. HC1 was added and the mixture was allowed to stand for 3 days at 37 °. The acid was removed by distillation under reduced pressure. The residue was suspended in IOO ml of distilled water and then taken to dryness again. Resuspension of the residue in distilled water and concentration of the solution was followed by transferring the mixture to a test tube and adjusting to pH 6. After centrifuging, the supernatant and the four 5-ml washings of the precipitate were placed in 5o-ml volumetric flasks and diluted to volume. This was a mixture of peptides from the partial hydrolysis of cytolyzate. A o.3-ml sample was completely hydrolyzed by adding 0.3 ml of conc. HC1 and hydrolysis was carried out in an oven at IOO° for 15 h. The excess acid was removed by evaporating to dryness in a vacuum desiccator over NaOH pellets. When dry, distilled water was added and the sample was taken to dryness again. Dilute NH4OH was added to neutralize the remaining acid and the mixture was again dried. The
wf~, t
\
Fig. 5. C h r o m a t o g r a p h i c s e p a r a t i o n of hydrolyzates of L. arabinosus cytolyzates. Left to right. Sample 1:2o ¢il s a m p l e of protein h y d r o l y z a t e of o r g a n i s m s g r o w n in the presence of F P A partially hydrolyzed and treated with p h e n y l i s o t h i o c y a n a t e prior to acidic total hydrolysis. The r e c t a n g u l a r area s h o w s the area, from 3 c o m p a n i o n c h r o m a t o g r a m s , cut for biological a s s a y and r e c h r o m a t o graphing, and the area in which m o s t of the F P A is located. Sample 2: Mixed c h r o m a t o g r a m of area eluted from sample t plus ~o iLg of FPA. Sanlple 3: C h r o m a t o g r a m of area eluted from sample t. Sample 4 : 3 Hg of FPA. The dotted outlines, the solid outlines and the filled-in areas represent increasing intensities of n i n h y d r i n reaction.
Re/erences p. 327.
I
!
0
1
© ;.Z '~° 4'
"2-~" 1
2
3
©
326
R . S . BAKER, J. E. JOHNSON, S.W. FOX
VOL. 2 8 (1958)
residue was suspended in 0.3 ml of distilled water and the F P A was determined by paper chromatography and biological assay. Table IV shows the results. To another o.3-ml sample, 0.3 ml of pyridine containing 43 mg of PTC was added. The reaction was carried out as described before and after removal of the excess pyridine and PTC with benzene, the remaining mixture was now totally hydrolyzed with an equal volume of conc. HC1 in an oven at ioo ° for 15 h. Removal and neutralization of the acid was performed as above and the F P A was determined b y paper chromatography and biological assay. Fig. 5 and Table IV give the results. The results shown in Table IV and Fig. 5 agree qualitatively with the results in Fig. I, i.e., that F P A appears in the proteins of L. arabinosus under these growth conditions. In addition, the quantitative data in Table IV show that amino-bound F P A fraction (60-80 % of total FPA) is greater than would be expected (lO-16 %) from the incomplete blocking of free FPA. Included in Table IV are the results of another experiment similar to the one just described. DISCUSSION
The data presented indicate the incorporation of p-fluorophenylalanine into proteins of Lactobadllus arabinosus. Such a result is consistent with the fact that proteolytic enzymes which are not required to deal with such residues in nature, can nevertheless catalyze reactions involving them 3°. The spatial allowances of an organized polymolecular system such as a cell might be expected to be less, however, than is true for an isolated enzyme acting on a single substrate in a laboratory experiment. The experiments and analyses demonstrate that this much deviation from normal synthesis of protein is easily possible. No alterations in protein concomitant with phenotypic variation were observed, inasmuch as no phenotypic variation was demonstrated. The conclusion that protein synthesis varies demonstrably was also reached from studies in the normal variation of protein and a designation of these as a Darwinian continuity of differing structures 31 c/. 32. The process described here differs in being accomplished artificially with an unnatural amino acid. Although the adjustment to tolerance of p-fluorop1~,~1~ lalanine involves a genotypic change, the same cannot be said for the stimulat~,~y response. In the present state of knowledge, it would seem most likely that development of FPA-tolerance involves a change in the genetic apparatus but that FPA-stimulation represents merely an alteration of protein synthesis within its normal limits without genie alteration. The evidence for the latter is principally the absence of a lag period in the enhancement of growth. Such interpretation is consistent with the belief that genie alteration requires a change in nature of the nucleic acid portiGn of an organism 33. Early recognition that protein synthesis is subject to variation can be attributed to MACALLUM1, who in 1926 said, " . . . proteins, composed as they are each of a variable number of amino acids and in variable proportions of these, cannot be predicated as having a uniform composition even absolutely in similar cells in the same organism, for thus mutation would never obtain in a species". MACALLUM'S insight leaves open the possibility that variation in proteins is more of a cause than an effect, a point of view which would find little support, today, but his reasoning as it applies to the possibility of variation in protein structure is now supported l~e/erences p. 327.
VOL. 2 8
(1958)
•-FLUOROPHENYLALANINE
IN PROTEINS
327
b y experiments and analysis. In the light of the data presented, one can visualize that some latitude in protein synthesis is possible without mutation, whereas other variations require mutation. SUMMARY F o r t h e earlier suggested i n c o r p o r a t i o n of p-fluorophenylalanine into t h e p r o t e i n s of Lactobacillus arabinosus f u r t h e r evidence h a s been obtained. T h e cytolyzate of cellular p r o d u c t s of culture in the presence of the u n n a t u r a l a m i n o acid h a v e been partially hydrolyzed, t r e a t e d w i t h phenyli s o t h i o c y a n a t e a n d t o t a l l y hydrolyzed. The a m i n o acid has been found and assessed in t h e h y d r o l y t i c p r o d u c t . T h e significance of these results h a s been discussed.
REFERENCES x A. B. MACALLUM, Physiol. Revs., 6 (I926) 317 . 2 E. ABDERHALDEN AND P. RONA, Z. physiol. Chem., 46 (1905) I79. s S. TAMURA, Z. physiol. Chem., 88 (1913) 19o. 4 K. DIRR, Z. physiol. Chem., 26o (1939) 65. 5 j. L. STOKESAND M. GUNNESS, J. Bacteriol., 52 (1946) I956 F. k . CSONKA, C. A. DENTON AND S. J. RINGEL, J. Biol. Chem., 169 (1947) 259. F. A. CSONKA, J. Nutrition, 42 (195 o) 443s F. A. CSONKA AND M. JONES, J. Nutrition, 46 (1952) 531. 9 N. A. N. RAO AND T. K. WADHWANI, J. Bacteriol., 72 (1956) 12. 10 j . L. ROSEDALE, Biochem. J., 16 (1922) 27. u j . L. ROSEDALE, Biochem. J., 22 (1928) 826. 12 R. J. BLOCK, J. Biol. Chem., 133 (194o) 71. is j . C. FREELAND AND E. F. GALE, Biochem. J., 41 (1947) 135. It E. WORK, Biochim. Biophys. Acta, 3 (1949) 400. 15 D. E. ATKINSON, S. MELVIN AND S. V ~ F o x , Arch. Biochem. Biophys., 31 (1951) 205. 16 H. M. DYER, J. Biol. Chem., 124 (1938) 519. IT F. SCHLENK AND J. A. TILLOTSON, J. Biol. Chem., 206 (1954) 687. 18 E. P. PAINTER, Chem. Revs., 28 (1941) 179;T. W. TUVE AND H. H. WILLIAMS, J. Am. Chem. Soc., 79 (1957) 583019 H. TARVER, in H. NEURATH AND K. BAILEY, The Proteins, Vol. I I B, Academic Press, Inc., N e w York, 1954, P- 1295. a0 M. FLING AND S. W. FOX, J . Biol. Chem., 16o (1945) 33521 j . T. HOLDEN AND E. E. SNELL, J. Biol. Chem., 178 (1949) 799. 82 R. S. BAKER, J. E. JOHNSON AND S. W. FOX, Federation Proc., 13 (1954) 178. 23 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 21 (1956) 592. 24 O. L. HOFFMANN, S. W. FOX AND M. W. BULLOCK, J. Biol. Chem., 196 (1952) 437. 25 L. D. WRIGHT AND H. R. SKEGGS, J . Biol. Chem., 159 (1945) 611. 2e K. A. KUIKEN, W. H. NORMAN,C. M. LYMAN, V. HALE AND L. BLOTTER,]. Biol. Chem., 151 (1943) 615. 2~ S. W. F o x , T. L. HURST AND K. F. ITSCHNER, J. Am. Chem. Soc., 73 (1951) 3573. 28 D. GRoss AND H. TARVER, J. Biol. Chem., 217 (1955) 169. 39 S. M. PARTRIDGE, Biochem. J., 44 (1949) 521. 30 E. L. BENNETT AND C. NIEMANN, J. Am. Chem. Soc., 72 (1950) 18oo. 31 S. W. F o x , Am. Scientist, 44" (1956) 34733 C. A. KNIGHT, Advances in Virus Research, 2 (1954) 153. 33 H. KACSER, Science, 124 (1956) 151.
Received November Ist, 1957
BIOCHIMICA ET BIOPHYSICA ACTA
INCORPORATION OF p-FLUOROPHENYLALANINE OF LACTOBACILLUS
VOL. 2 8 ( I 9 5 ~ )
INTO PROTEINS
ARABINOSUS*
R. S. B A K E R * * , J O S E P H E. J O H N S O N * * * AND S I D N E Y XV. FOX§
Chemical Laboratory, Iowa State College, Ames, Iowa (U.S.,4,)
The concept that the phenotypic expression of an organism may be correlated with the identity of its constituent proteins, has been in the literature for decades 1. Any alteration in the content of any one amino acid in a protein molecule might accordingly be related to a detectable alteration in the organism. A number of attempts to alter constitution of total proteins by control of the diet or medium have been recorded ~-s. Some of the claims of successful alteration of this kind have been refuted 1°-~4. The reported findings fail to meet the stated experimental objective for one or two main reasons: (I) only inadequately precise methods of amino acid assay were available for use and (2) a change in amino acid composition of an entire organism or tissue may be explained simply as a shift in proportion of proteins rather than as an alteration in composition of one or more proteins. Accordingly, the use of amino acids containing groups not found naturally becomes of interest. To the extent that it might be established that such an unnatural amino acid is incorporated into protein ~5, compelling evidence of the alterability of protein synthesis would be at hand. A substance which early received similar study is found in the work of DYER~6. DYER synthesized the now highly useful methionine antagonist, ethionine, and fed it to rats. She discovered thereby an early antimetabolite. Evidence for incorporation of ethionine into protein has been presented ss. Parenthetically, it may be noted that the sulfur of ethionine is metabolically available ~v. The suggestion of incorporation of selenocystine into protein has been offered ~8. Selenocystine may however be looked upon as a natural amino acid. The examples given indeed indicate that there is not an a priori qualitative distinction between natural amino acids and unnatural amino acids. The deciding factors may be merely the identities of the amino acids in the environment and the opportunity for the organism to adjust to them. Results most easily interpreted as incorporation of an unnatural or unusual amino acid were reported with p-fluorophenylalanine (FPA) in 195 I15. This appears to * J o u r n a l P a p e r No. J-3284 of the I o w a Agricultural and H o m e Economics E x p e r i m e n t Station, Ames, Iowa. Project t [ Tt, s u p p o r t e d in p a r t by the Rockefeller Foundation. F r o m the M.S. thesis (i951) and the P h . D . thesis (1953) of R. S. BAKER. ** Present address: Eli Lilly and Co., Indianapolis, Ind. * * * Present address: Chemistry 1)epartment, Massachusetts I n s t i t u t e of Technology, Cambridge, Mass. § Present address: C h e m i s t r y l ) e p a r t m e n t and Oceanographic Institute, l'lorida State University, Tallahassee, Fla.
]{e[erences p, 327.
VOL. 2 8
(1958)
p-FLUOROPHENYLALANINE IN PROTEINS
319
be the first recorded instance of the incorporation of an unnatural amino acid into the protein of an organism ~9. An earlier, doubtful possibility of the incorporation of D-alanine ~° has been shown to involve an amino acid which is definitely natural in its n form2h More conclusive evidence of incorporation of FPA into proteins of L. arabinosus was presented in 195 4 by way of partial hydrolysis followed by blocking N-terminal and free amino acids with phenylisothiocyanate and total hydrolysis of the peptides remaining 22. These experiments and subsequent experiments are described in detail in this paper. MUNIER AND COHN23 have recently reported the incorporation of p-fluorophenylalanine, and of thienylalanine, into the proteins of Escherichia coll.
Besides the unlikelihood of its natural occurrence, p-fluorophenylalanine was selected for the present study because of the small halogen atom and the consequent similarity of FPA to the aromatic amino acids phenylalanine and tyrosine, because of the unlikelihood of transfer of a w l fluoride to another amino acid 24, and because of the presumed feasibility of selectively following fluorine by elemental analysis, in contrast to determining altered ratios of natural amino acids in proteins. In practice the high RF of F P A in the solvents employed proved to be more useful in tracing it; in a developing solvent of tert.-butyl a l c o h o l - m e t h y l ethyl k e t o n e - w a t e r (4: 4: 1.5), FPA ran ahead of all amino acids in protein hydrolyzates.
METHODS
Organisms Laetobacillus arabinosus (ATCC 8o14) w a s m a i n t a i n e d as p r e v i o u s l y de s c ri be d, as w e re p h e + t y r +, t y r +, a n d t h e F P A - t o l e r a n t s t r a i n 15. L. arabinosus \ V r i g h t w a s a bl e t o s y n t h e s i z e its t r y p t o p h a n ; it w a s o b t a i n e d f r o m Dr. L. D. WRIGHT 25. I t di d n o t lose i t s a b i l i t y t o s y n t h e s i z e tryptophan when the stock culture was transferred.
Culture o[ organisms The b a s a l m e d i u m of KUIKEN et al. ze w a s used w i t h t h e p r o p e r a m o u n t s of P A a n d F P A a d d e d . F o r p l a t e a s s a y s , w a s h e d a g a r w a s a d d e d (I3/4 %). The m e d i u m w a s a n t o c l a v e d for 15-2o m i n a t 15 lb. p r e s s u r e a n d i n c u b a t e d a t 3 6 - 3 7 °. G r o w t h w a s m e a s u r e d in IO ml c u l t u r e s in o p t i c a l l y m a t c h e d P y r e x t u b e s ( i i x 15 ° ram) w i t h a Model I i C o l e m a n U n i v e r s a l s p e c t r o p h o t o m e t e r , a t 575 mtt.
Preparation o[ cy/olyzate \Vhen g r o w t h h a d ceased, t h e cells were collected b y c e n t r i f u g a t i o n iu a S h a r p l e s c o n t i n u o u s c e n t r i f u g e . The cells were s u s p e n d e d in p h y s i o l o g i c a l s a l i n e o v e r n i g h t . A f t e r c e n t r i f u g i n g , t h e cells were r e s u s p e n d e d in saline a n d c e n t r i f u g e d agai n. The cells w e r e c y t o l y z e d b y g r i n d i n g w i t h s a n d a n d e t h e r ; t h e e t h e r w a s d e c a n t e d a n d e x t r a c t e d s e v e r a l t i m e s w i t h w a t e r . The a q u e o u s e x t r a c t s were c o m b i n e d w i t h t h e c y t o l y z a t e .
Blocking o[ [ree amino acids N i t r o g e n w a s d e t e r m i n e d in t h e c y t o l y z a t e b y m i c r o - K j e l d a h l , a n d p h e n y l i s o t h i o c y a u a t o (PTC) w a s a d d e d 27 on t h e b a s i s t h a t o n e - h a l f of t h e n i t r o g e n w a s p r e s e n t as flee a m i n o g r o u p s w i t h an a d d i t i o n a l n i n e t e e n - f o l d excess. An e q u a l v o l u m e of p y r i d i n e w a s a d d e d t o an a l i q u o t of t h e c y t o l y z a t e a n d t h e m i x t u r e w a s m a d e a l k a l i n e to b r o m o t h v m o l bl ue w i t h N a O H . The PTC w a s a d d e d a n d t h e r e a c t i o n k e p t a l k a l i n e b y t h e p e r i o d i c a d d i t i o n of N a O H w h i l e t h e r e a c t i o n w a s i n c u b a t e d a t 37 °. \Vhen t h e c o n s u m p t i o n of a l k a l i ceased, t h e excess p y r i d i n e a u d PTC w e r e r e m o v e d b y e x t r a c t i o n w i t h five v o l u m e s of benzene. The r e m a i n i n g m i x t u r e w a s h y d r o l y z e d w i t h an e q u a l v o l u m e of cone. HCl.
Paper chromatography P a p e r c h r o m a t o g r a m s were rim on \ V h a t m a n No. t filter p a p e r in s h e e t s of 7 i~ "~ q 14 i nc he s in c h a m l ) e r s which were .5-11). c h e m i c a l - r e a g e n t b o t t l e s w i t h s c r e w caps. A c y l i n d e r w a s m a d e of t h e p a p e r b y s t a p l i n g t h e t w o o p p o s i t e edges t o g e t h e r w i t h o u t t h e e dge s t o u c h i n g . F or o p t i u l u n l
Re#'rences p. 327.
320
R.S.
BAKER,
J. E. JOHNSON,
S. W . F O X
VOL. 28
(I958)
s e p a r a t i o n of F P A a n d P A t h e s o l v e n t of t e r l . - b u t y l a l c o h o l - m e t h y l e t h y l k e t o n e - w a t e r ( 4 : 4 : 1 . 5 b y v o l u m e ) w a s r u n to t h e t o p of t h e p a p e r 3 t i m e s w i t h t h e c y l i n d e r r e m o v e d f r o m t h e c h a m b e r a n d dried b e t w e e n each a s c e n t to t h e top. F or q u a n t i t a t i v e r e s u l t s t h e p a p e r c h r o m a t o g r a m w a s d i p p e d i n t o o.2 % n i n h y d r i n in a c e t o n e a n d a l l o w e d t o d r y in a i r a t room t e m p e r a t u r e . E s t i m a t i o n w a s m a d e b y v i s u a l c o m p a r i s o n of color i n t e n s i t i e s of u n k n o w n s a m p l e s with standards. Inhibition assay
T h e zone of i n h i b i t i o n c a u s e d b y F P A on L. a r a b i n o s u s p h e + t y r + w a s c o m p a r e d w i t h t h e a r e a of i n h i b i t i o n of t h e u n k n o w n s a m p l e s . The F P A w a s a d d e d t o t h e a g a r p l a t e b y p l a c i n g t h e filter p a p e r d i s k c u t f r o m a c h r o m a t o g r a m o n t o t h e p r e v i o u s l y i n o c u l a t e d a n d solidified m e d i u m . P h e n y l a l a n i n e a n d t y r o s i n e were o m i t t e d from t h e m e d i u m for t h i s o r g a n i s m . The p a p e r d i s k c o n t a i n i n g t h e F P A w a s c u t on t h e b a s i s of t h e l o c a t i o n of F P A on a c o m p a n i o n c h r o m a t o g r a m w h i c h h a d been t r e a t e d w i t h n i n h y d r i n . The a r e a of i n h i b i t i o n w a s r e l a t e d to t h e a m o u n t of L - F P A *. RESULTS
The RF values of phenylalanine, p-chlorophenylalanine, and FPA are given in Table I. It is seen that each of the halophenylalanines travels ahead of phenylalanine, which is the fastest of the amino acids from protein hydrolyzates, in this solvent mixture. Of the two unnatural amino acids, the chloro analog exhibits the higher RF. In Fig. • are presented the results of a standard assay of FPA by inhibition analysis. A fresh standard was prepared for each estimation. In each case, the plot tended to be linear in the range in which values had meaning in the experiments. TABLE
I
~ F VALUES OF P H E N Y L A L A N I N E , p - C H L O R O P H E N Y L A L A N I N E , AND p - F L U O R O P H E N Y L A L A N I N E IN /e~'/.-BUTYL ALCOHOL METHYL ETHYL K E T O N E - - W A T E R ( 4 : 4 : i . 5 ) A m i n o acid
RF
Phenylalanine p-Chlorophenylalanine p-Fluorophenylalanine
i
15-
~
0.60 0.84 o. 76
,
•
,z O
ilJ o o 0 ]" 1100
I I ; 1200 1300 1400 AREA OF INHIBITION IN SQUARE M I L L I M E T E R S
1500
Fig. r. P l o t of a r e a of i n h i b i t i o n in d i s k a s s a y vs. e o n c e n t r a t i c m of ~r_-/~-fluorophenylalanine. * O t h e r e x p e r i m e n t s w i t h k n o w n a m o m l t s of free F P A m i x e d w i t h a p o r t i o n of t h e c y t o l y z a t e of o r g a n i s u l s g r o w n in t h e a b s e n c e of F I ' A s h o w e d t h a t 85-90 % of t h e free F P A could be b l o c k e d by this method. l¢e/ereJ*ces p. 327 .
VOL. 28 (1958)
p-FLUOROPHENYL\L\NINE
321
IN PROTEINS
60
S
2/
70-
¢ 80-
90-
/ 100 -
~__~z 10
/
I I ! I I I I ! I ! t
I
n I
O"F'
20
30
40
INCUBATION
TIME
J 5O
60
"to
80
(HOURS)
Fig. 2. L. arabinosus g r o w n in m e d i u m c o n t a i n i n g a i o : i ratio of p - f l u o r o p h e n y l a l a n i n e to p h e n y l alanine. T y p i c a l t i m e - g r o w t h r e l a t i o n s h i p of L. arabinosus on c u l t u r i n g (solid curve) a n d s u b c u l t u r i n g ( d o t t e d curve) in t h e presence of p - f l u o r o p h e n y l a l a n i n e , o.o8 t, moles p h e n y l a l a n i n e per t u b e , o.8 t, moles p - f l u o r o p h e n y l a l a n i n e p e r tube.
In Fig. 2 are presented the results of subculturing parental strain in the presence of an inhibitory proportion of F P A (IO F P A : I PA on a DL molar basis). It is clear t h a t the parental strain grows only after an extended lag period. Once the surviving adjusted cells are reinoculated into medium containing F P A at the parentally inhibitory level, initiation of growth is as rapid as in the controls. This evidence substantiates the conclusion, based on the phenomenon of haphazard appearance of F P A tolerance 15, that the tolerant strain is a naturally selected m u t a n t . These results are for FPA-tolerance. The phenomenon of stimulation b y subinhibitory ratios of F P A has exhibited no time lag in a n y of the numerous trials. No evidence is at h a n d t h a t stimulation of the parental strain involves mutation, nor is there any evidence for the puzzling interpretation b y others t h a t the organism develops a requirement for the analog in these experiments 2s. On the contrary, the early evidence was interpretable as direct utilization of F P A b y the parental strain 15. "Fable I I reveals trends which have been observed also in a n u m b e r of other experiments. The FPA-tolerant strain is virtually unaffected b y growth in subinhibitory concentrations of FPA. The parental strain however demonstrates stiinulation. The difference between these two was larger in other trials. The other three strains of L. arabinosus suffered marked inhibition at this level. Tile sensitivity to inhibition b y F P A b y three of the strains also explains w h y one of these, t)he + tyr+, was selected for use in inhibition assays. Table I I I represents typical stimulation due to F P A at limiting concentrations of PA. This type of result was obtained repeatedly. Tables I I I and IV present the results of large scale stimulation experiments. 111 a typical one of these, 3 ° 1 of medium containing 39.6 mg I)L-PA (phenylalaifine) and 98.6 mg 1)L-FPA, was harvested after 48 h of incubation of L. arahim~szls at 3T'The cells were cytolyzed; to the final vohituc of 3oo ml an equal voluine ol pyridine aud 5 ° g of PTC was added. The reaction was considered to be complete when consumption of alkali had stopped; the solvent was then removed by distillation under reduced pressure. The residue was resuspended in 1oo ml of distilled water which Re/erences p. ,72 7.
322
R.S.
B A K E R , J . E. J O H N S O N , S. W . I;OX TABLE
VOL.
FPA/PA molar ratio
(1058)
II
GROWTH RESPONSE OF VARIOUS STRAINS OF L a c l o b a c i l l u s TO p-FLUOROPHENYLALANINE *
Strain
28
arabinosus
Incubation time (hours) 7
x6
24
37
4°
48
55
Parental
o 2.5
91 92
62 69
59 53
56 49
-44
-44
---
Tyr +
o 2.5
93 --
57 --
47 95
4° 94
34 83
31 71
-65
Phe + tyr +
o 2.5
92 --
42 --
21 91
18 88
16 72
--61
-54
Wright
o 2.5
92 93
8o 86
63 81
61 80
-81
-80
-77
FPA-Tolerant
o 2-5
98 97
86 83
66 63
60 58
60 56
---
---
PA =
phenylalanine,
FPA
-- fluorophenylalanine
* V a l u e s a r e g i v e n in p e r c e n t t r a n s m i s s i o n , a n d a r e a v e r a g e s o f q u a d r u p l i c a t e D L - P A p r e s e n t is a t l e v e l o f o . o 8 / * m o l e p e r t u b e . TABLE GROWTH RESPONSE OF L . a r a b i n o s u s
UNDER LIMITING CONDITIONS
Incubation time in hours
nL-FPA/tube*
24
31
48
o 0.20
56 5°
54 46
53 45
PA = * Each tube contains duplicate io-ml cultures.
III
TO F P A
Micromoles o/
phenylalanine,
0.08/,mole
io-ml cultures
FPA
=
fluorophenylalanine
of D L - P A . V a l u e s a r e p e r c e n t t r a n s m i s s i o n ;
TABLE
averages
of
IV
BIOLOGICAL ASSAY FOR TOTAL AND AMINO-BOUND F P A IN ALIQUOTS OF L . a r a b i n o s u s CYTOLYZATE SUBJECTED TO PARTIAL HYDROLYSIS, P r C TREATMENT, AND TOTAL HYDROLYSIS Culturist
Assayist
Total FPAI20 I~l
RSB RSB MM
RSB JEJ JEJ
IO /*g* 15 (16.2, 15.5, 14.5) 15 ( 1 5 . i , 15.6 )
Amino-bound FPA/20 Id 8 /*g* i o (lO.6, ~o.6, 2o.6, 9.8) i i (12.o, l I . 2 , IO.6)
* By paper-chromatographic methods, i2/*g FPA/2o/*1 were found for the total FPA and 6 / * g F P A / 2 o / * 1 f o r t h e a m i n o - b o u n d F P A . F P A -- p - f l u o r o p h e n y l a l a n i n e , PTC = phenylisothiocyanate.
was then r e m o v e d u n d e r r e d u c e d pressure. T h e residue was h y d r o l y z e d b y s u s p e n d i n g in 2oo ml of distilled w a t e r a n d 2oo ml of conc. HC1 a n d refluxing for 24 h. A f t e r t h e acid was r e m o v e d u n d e r r e d u c e d pressure, the residue was again s u s p e n d e d in distilled w a t e r a n d t a k e n to dryness again. The residue was e x t r a c t e d with 25o ml of d i e t h y l ether a n d 2oo ml of distilled water. The e t h e r l a y e r was s e p a r a t e d a n d w a s h e d with two 5o-ml p o r t i o n s of distilled water. T h e e t h e r washings were a d d e d ReJerences p. 327,
VOL. 2 8 (I(~58)
p-FLUOR()PHENYLALANINE IN PROTEINS
323
aho
O
QO
[bO dZT-'o
Fig. 3. Chromatographic analysis of hydrolyzate of L. a r a b i n o s u s . Left to right. Phenylalanine; Phenylalanine and p-fluorophenylalanine; Sample F, (see text); Sample F and p-fluorophenylalanine; pfluorophenylalanine; Sample I (see text); Sample I and added p-fluorophenylalanine. The dotted lines, the solid lines, and the filled in areas represent the increasing intensity of ninhydrin reacting areas.
O
c=3 PA
PA FPA
F
o F and FPA
FPA
I
O I and FPA
to the residue remaining in the aqueous layer of the extraction procedure. Two and one-half g of activated Norite A ~9was added to the aqueous solution and the mixture was shaken mechanically for 4 h. The filtered charcoal was eluted by stirring with 250 ml of 20 % aqueous acid containing 5 % phenol. After the charcoal was filtered, the filtrate was extracted with 300 ml of diethyl ether to remove the excess phenol and the ether extract was washed with ioo ml of distilled water which was combined with the filtrate and taken to dryness under reduced pressure. The residue was suspended in 50 ml of distilled water, centrifuged, the supernatant was poured off, and the residue was resuspended in distilled water and centrifuged again. The supernatants were combined and concentrated under reduced pressure. The precipitate which formed was centrifuged, washed with a small quantity of hot distilled water, the supernatant was combined with the concentrated solution and the pH was adjusted to 6 with 0.2 N NaOH. On the chromatograms in Figs. 3 and 4 this is sample F. Two controls were employed to determine the efficiency of the PTC treatment for the removal of free FPA .and to make certain the FPA could be detected if present in the protein hydrolyzate. For these controls, L. arabinosus cells grown on a complete synthetic medium without FPA were used and the procedure was the same as used in the previous experiment. In control G, 8 mg of DL-FPA was added to the cytolyzate before the treatment with PTC in order to see if the treatment blocked the free amino acids. In control sample H, 8 mg of DL-FPA was added after PTC treatment in order to ascertain if FPA, which was known to be present, could be detected. Control I did not contain any FPA. The results are shown in the chromatograms in Figs. 3 and 4. Ill Fig. 3, the chromatogram shows the following: (i) PA and FPA are readily separated, (ii) sample F contains a ninhydrin spot which has the same RF as FPA and is intensified R e / e r e n c e s p. 327 .
3°-4
R.s.
Ik\I,iER, J. E. JOHNSON, S. W. FOX
Q e
VOL. 2 8 (I05(~)
o
0 o
©o
0
£.
~
.;
,2-2,
i t
t
' ,
'i i:Z25 ! : Q)
i i
6
c~
0
xCJ PA
G
H
FPA
F
I
FPA
Fig. 4. C h r o m a t o g r a p h i c s e p a r a t i o n of h y d r o l y z a t e s of s a m p I e s F, G, H, a n d I. L e f t to right. P h e n y l a l a n i n e ; S a m p l e G (see text) ; S a m p l e H (see text) ; p - P l u o r o p h e n y l a l a n i n e ; S a m p l e F (see t e x t ) ; S a m p l e I (see t e x t ) ; p - F l u o r o p h e n y l a l a nine. T h e d o t t e d lines, t h e solid lines a n d t h e enclosed a r e a s r e p r e s e n t a r e a s of increasing intensities of n i n h y d r i n reaction.
PA
when F P A is added to sample F. A similar ninhydrin spot does not result in control I unless F P A is added to it. In Fig. 4, the c h r o m a t o g r a m shows the following : (i) control sample G does not show a ninhydrin spot for F P A indicating that PTC treatment removed the free F P A added to the extent that it can be determined b y qualitative paper-chromatographic methods, (ii) control sample H shows the presence of FPA, indicating that the acid hydrolysis and subsequent procedures do not cause an appreciable loss of,FPA, (iii) sample F shows the presence of F P A and control sample I does not show F P A . In order to assess the quantitative extent of the blocking of free F P A b y PTC 2.2i m g o t I)I,-FPA were treated with PTC and hydrolyzed according to the procedures outlined above. After removal of the acid, the residue was suspended in distilled water and taken to dryness. The residue was suspended in o.3 ml of distilled water and brought to p H 5 with o.4 ml of o.o5 N N a O H and assayed. After chromatographing the material with standard quantities of FPA, it was estimated that lO% of the F P A was not blocked b y PTC treatment. A second experiment was set up like control sample (i except that 30 mg of I)L-FPA was added to a cytolyzate preparation from L. arabinosus grown in a complete medium without FPA. This was treated with PTC, hydrolyzed, and extracted with ether as before and the mixture diluted to 5 ° ml and used for assay. Charcoal absorption and elution was omitted. Chromatograms failed to resolve the amino acids sufficiently for a quantitative comparison of ninhydrin colors with F P A standards; accordingly, bioassay was used. This assay indicated that I0°{, of the free F P A was not blocked by PTC treatment. IDI~NTIFIC.VI'ION OF AMINO-I~OUNI~ F P A IN PEPTIDES FROM L. arali.noslts
The experiments on the blocking of free F P A indicate that the blocking was 8 4 - 9 o % Re/erences p. 327.
VOL. 2 ~ ( I 9 5 8 )
p-FLUOROPHENYL.KLANINE IN PROTEINS
325
complete. To determine if the amount of F P A found in the cytolyzate of ceils grown under stimulating conditions was more or less than could be attributed to incomplete blocking of free FPA, quantitative estimations of the total F P A and the amino-bound F P A were made on peptides obtained by the partial hydrolysis of the cytolyzate. The partial hydrolysis presumably also served to liberate free amino acids trapped in proteins, but not in peptide linkage. After incubation in 3o 1 of medium containing 39.6 mg DL-PA, and 98.9 mg DI~-FPA for 48 h the L. arabinosus cells were harvested. Growth was followed in Io-ml cultures containing the same medium and the same medium without FPA. The cells were cytolyzed as described above and to the 25o-ml volume, 250 ml of conc. HC1 was added and the mixture was allowed to stand for 3 days at 37 °. The acid was removed by distillation under reduced pressure. The residue was suspended in IOO ml of distilled water and then taken to dryness again. Resuspension of the residue in distilled water and concentration of the solution was followed by transferring the mixture to a test tube and adjusting to pH 6. After centrifuging, the supernatant and the four 5-ml washings of the precipitate were placed in 5o-ml volumetric flasks and diluted to volume. This was a mixture of peptides from the partial hydrolysis of cytolyzate. A o.3-ml sample was completely hydrolyzed by adding 0.3 ml of conc. HC1 and hydrolysis was carried out in an oven at IOO° for 15 h. The excess acid was removed by evaporating to dryness in a vacuum desiccator over NaOH pellets. When dry, distilled water was added and the sample was taken to dryness again. Dilute NH4OH was added to neutralize the remaining acid and the mixture was again dried. The
wf~, t
\
Fig. 5. C h r o m a t o g r a p h i c s e p a r a t i o n of hydrolyzates of L. arabinosus cytolyzates. Left to right. Sample 1:2o ¢il s a m p l e of protein h y d r o l y z a t e of o r g a n i s m s g r o w n in the presence of F P A partially hydrolyzed and treated with p h e n y l i s o t h i o c y a n a t e prior to acidic total hydrolysis. The r e c t a n g u l a r area s h o w s the area, from 3 c o m p a n i o n c h r o m a t o g r a m s , cut for biological a s s a y and r e c h r o m a t o graphing, and the area in which m o s t of the F P A is located. Sample 2: Mixed c h r o m a t o g r a m of area eluted from sample t plus ~o iLg of FPA. Sanlple 3: C h r o m a t o g r a m of area eluted from sample t. Sample 4 : 3 Hg of FPA. The dotted outlines, the solid outlines and the filled-in areas represent increasing intensities of n i n h y d r i n reaction.
Re/erences p. 327.
I
!
0
1
© ;.Z '~° 4'
"2-~" 1
2
3
©
326
R . S . BAKER, J. E. JOHNSON, S.W. FOX
VOL. 2 8 (1958)
residue was suspended in 0.3 ml of distilled water and the F P A was determined by paper chromatography and biological assay. Table IV shows the results. To another o.3-ml sample, 0.3 ml of pyridine containing 43 mg of PTC was added. The reaction was carried out as described before and after removal of the excess pyridine and PTC with benzene, the remaining mixture was now totally hydrolyzed with an equal volume of conc. HC1 in an oven at ioo ° for 15 h. Removal and neutralization of the acid was performed as above and the F P A was determined b y paper chromatography and biological assay. Fig. 5 and Table IV give the results. The results shown in Table IV and Fig. 5 agree qualitatively with the results in Fig. I, i.e., that F P A appears in the proteins of L. arabinosus under these growth conditions. In addition, the quantitative data in Table IV show that amino-bound F P A fraction (60-80 % of total FPA) is greater than would be expected (lO-16 %) from the incomplete blocking of free FPA. Included in Table IV are the results of another experiment similar to the one just described. DISCUSSION
The data presented indicate the incorporation of p-fluorophenylalanine into proteins of Lactobadllus arabinosus. Such a result is consistent with the fact that proteolytic enzymes which are not required to deal with such residues in nature, can nevertheless catalyze reactions involving them 3°. The spatial allowances of an organized polymolecular system such as a cell might be expected to be less, however, than is true for an isolated enzyme acting on a single substrate in a laboratory experiment. The experiments and analyses demonstrate that this much deviation from normal synthesis of protein is easily possible. No alterations in protein concomitant with phenotypic variation were observed, inasmuch as no phenotypic variation was demonstrated. The conclusion that protein synthesis varies demonstrably was also reached from studies in the normal variation of protein and a designation of these as a Darwinian continuity of differing structures 31 c/. 32. The process described here differs in being accomplished artificially with an unnatural amino acid. Although the adjustment to tolerance of p-fluorop1~,~1~ lalanine involves a genotypic change, the same cannot be said for the stimulat~,~y response. In the present state of knowledge, it would seem most likely that development of FPA-tolerance involves a change in the genetic apparatus but that FPA-stimulation represents merely an alteration of protein synthesis within its normal limits without genie alteration. The evidence for the latter is principally the absence of a lag period in the enhancement of growth. Such interpretation is consistent with the belief that genie alteration requires a change in nature of the nucleic acid portiGn of an organism 33. Early recognition that protein synthesis is subject to variation can be attributed to MACALLUM1, who in 1926 said, " . . . proteins, composed as they are each of a variable number of amino acids and in variable proportions of these, cannot be predicated as having a uniform composition even absolutely in similar cells in the same organism, for thus mutation would never obtain in a species". MACALLUM'S insight leaves open the possibility that variation in proteins is more of a cause than an effect, a point of view which would find little support, today, but his reasoning as it applies to the possibility of variation in protein structure is now supported l~e/erences p. 327.
VOL. 2 8
(1958)
•-FLUOROPHENYLALANINE
IN PROTEINS
327
b y experiments and analysis. In the light of the data presented, one can visualize that some latitude in protein synthesis is possible without mutation, whereas other variations require mutation. SUMMARY F o r t h e earlier suggested i n c o r p o r a t i o n of p-fluorophenylalanine into t h e p r o t e i n s of Lactobacillus arabinosus f u r t h e r evidence h a s been obtained. T h e cytolyzate of cellular p r o d u c t s of culture in the presence of the u n n a t u r a l a m i n o acid h a v e been partially hydrolyzed, t r e a t e d w i t h phenyli s o t h i o c y a n a t e a n d t o t a l l y hydrolyzed. The a m i n o acid has been found and assessed in t h e h y d r o l y t i c p r o d u c t . T h e significance of these results h a s been discussed.
REFERENCES x A. B. MACALLUM, Physiol. Revs., 6 (I926) 317 . 2 E. ABDERHALDEN AND P. RONA, Z. physiol. Chem., 46 (1905) I79. s S. TAMURA, Z. physiol. Chem., 88 (1913) 19o. 4 K. DIRR, Z. physiol. Chem., 26o (1939) 65. 5 j. L. STOKESAND M. GUNNESS, J. Bacteriol., 52 (1946) I956 F. k . CSONKA, C. A. DENTON AND S. J. RINGEL, J. Biol. Chem., 169 (1947) 259. F. A. CSONKA, J. Nutrition, 42 (195 o) 443s F. A. CSONKA AND M. JONES, J. Nutrition, 46 (1952) 531. 9 N. A. N. RAO AND T. K. WADHWANI, J. Bacteriol., 72 (1956) 12. 10 j . L. ROSEDALE, Biochem. J., 16 (1922) 27. u j . L. ROSEDALE, Biochem. J., 22 (1928) 826. 12 R. J. BLOCK, J. Biol. Chem., 133 (194o) 71. is j . C. FREELAND AND E. F. GALE, Biochem. J., 41 (1947) 135. It E. WORK, Biochim. Biophys. Acta, 3 (1949) 400. 15 D. E. ATKINSON, S. MELVIN AND S. V ~ F o x , Arch. Biochem. Biophys., 31 (1951) 205. 16 H. M. DYER, J. Biol. Chem., 124 (1938) 519. IT F. SCHLENK AND J. A. TILLOTSON, J. Biol. Chem., 206 (1954) 687. 18 E. P. PAINTER, Chem. Revs., 28 (1941) 179;T. W. TUVE AND H. H. WILLIAMS, J. Am. Chem. Soc., 79 (1957) 583019 H. TARVER, in H. NEURATH AND K. BAILEY, The Proteins, Vol. I I B, Academic Press, Inc., N e w York, 1954, P- 1295. a0 M. FLING AND S. W. FOX, J . Biol. Chem., 16o (1945) 33521 j . T. HOLDEN AND E. E. SNELL, J. Biol. Chem., 178 (1949) 799. 82 R. S. BAKER, J. E. JOHNSON AND S. W. FOX, Federation Proc., 13 (1954) 178. 23 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 21 (1956) 592. 24 O. L. HOFFMANN, S. W. FOX AND M. W. BULLOCK, J. Biol. Chem., 196 (1952) 437. 25 L. D. WRIGHT AND H. R. SKEGGS, J . Biol. Chem., 159 (1945) 611. 2e K. A. KUIKEN, W. H. NORMAN,C. M. LYMAN, V. HALE AND L. BLOTTER,]. Biol. Chem., 151 (1943) 615. 2~ S. W. F o x , T. L. HURST AND K. F. ITSCHNER, J. Am. Chem. Soc., 73 (1951) 3573. 28 D. GRoss AND H. TARVER, J. Biol. Chem., 217 (1955) 169. 39 S. M. PARTRIDGE, Biochem. J., 44 (1949) 521. 30 E. L. BENNETT AND C. NIEMANN, J. Am. Chem. Soc., 72 (1950) 18oo. 31 S. W. F o x , Am. Scientist, 44" (1956) 34733 C. A. KNIGHT, Advances in Virus Research, 2 (1954) 153. 33 H. KACSER, Science, 124 (1956) 151.
Received November Ist, 1957