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The incorporation of p-fluorophenylalanine into some rabbit enzymes and other proteins
BIOCHIMICA ET BIOPHYSICA ACTA
THE INCORPORATION OF p-FLUOROPHENYLALANINE
145
INTO
SOME R A B B I T E N Z Y M E S A N D O T H E R P R O T E I N S E. W. W E S T H E A D * AND P. D. B O Y E R
Department o[ Physiological Chemistry, University of Minnesota, Minneapolis, Minn. (U.S.A.) (Received March 2oth, 1961)
SUMMARY
p-Fluorophenyl-E3-14Clalanine, when fed to rabbits, is incorporated into proteins of muscle, blood, and liver. The incorporation is extensive and takes place to approximately the same extent among proteins fractionated in a variety of ways. The p-fluorophenylalanine does not substitute for tyrosine to any measurable extent and is presumed to replace phenylalanine. Aldolase with a replacement of 25 % and glyceraldehyde 3-phosphate dehydrogenase with a replacement of approx. 16 % of the phenylalanine residues were crystallized in good yields under the same conditions used for the normal enzymes. Enzymic activities of the crystallized p-fluorophenylalanine-containing enzymes were the same as those of normal enzymes. The aldolase was chromatographically homogeneous with respect to p-fluorophenylalanine content and specific activity. Heat denaturation of glyceraldehyde 3-phosphate dehydrogenase showed that there was no detectable difference between the stabilities of the normal enzyme and the p-fiuorophenylalanine-containing enzyme.
INTRODUC~ON
Considerable evidence has accrued that the protein synthesizing mechanisms of bacteria do not show an absolute specificity for the natural amino acids. For example, 7-azatryptophan 1 and tryptazane ~ may substitute for tryptophan; p-fluorophenylalanine 3-7 and fl-2-thienylalanine 5 may substitute for phenylalanine; norleucine 5, selenomethionine s, and ethionine 9,1° may substitute for methionine; and canavanine may substitute for arginine n. In contrast, meager data are available concerning amino acid analog incorporation by mammalian systems. Reports from studies with intact animals appear to be limited to the demonstration of incorporation of norleucine into casein by cows 12 and the preliminary report 13 of the studies given in more detail herein demonstrating incorporation of p-fluorophenylalanine into rabbit proteins. I n vitro studies have shown incorporation of p-fluorophenylalanine into lysozyme and ovalbumin by minced hen oviducO 4 and into hemoglobin by erythrocytes x~, of canavanine into tumor cells in tissue culture 16, and of lysine analogs by rat-bone marrow preparationslL * Present address: D e p a r t m e n t of Biochemistry, D a r t m o u t h Medical School, Hanover, N.H.
(U.S.A.). Biochim. Biophys. Acta, 54 (1961) I 4 5 - I 5 ~
140
E. W. WESTFIEAD, P. D. BOYER
Information from and implications of studies relating to the specificity of protein synthesis have recently been ably reviewed by VAUGI~AN AND STEINBERGis. Recent information on this topic is also included in a review by COHENAND GROS19. A question of considerable importance is to what extent the incorporation of an amino acid analog may modify the molecular and functional properties of a particular protein. Evidence from bacterial studies indicates that in presence of some analogs proteins are synthesized with a modified composition but which are catalytically activO s. Further, crystalline B. subtilis a-amylase containing considerable ethionine retains full catalytic activity 1°. Replacement of about 1/6 of the phenylalanine by p-fluorophenylalanine, however, gave an amylase of the same physical properties but only about 7o % of the normal activity 6, and incorporation into penicillinase resulted in extensive activity lossL The studies reported herein were undertaken to find if p-fluorophenylalanine would be incorporated into proteins of the intact animal, and, if so, what readily detectable changes in properties of some proteins might result. EXPERIMENTAL
Materials and chemicals p-Fluoro-DL-phenylalanine was used as obtained from California Biochemical Corporation, Los Angeles 63, California. p-Fluoro-DL-phenylalanine labeled with 14C in the/~ position with an activity of 2.35 mC/mmole was obtained from the French Atomic Energy Commission. The recorded ultraviolet absorption spectrum of this material was identical with that of the California Biochemical Corporation product. Radioactive p-fluorophenylalanine was diluted with non-labeled material before addition to the diet. DEAE cellulose was purchased from Brown Mills, Keene, New Hampshire and washed with I M NaOH until colorless, before use. Ethanolized cellulose~o for column electrophoresis was purchased from A. B. Munktells, Grycksbo, Sweden. Phosphorylated cellulose used for aldolase chromatography was prepared by adding 35 g of Munktell's cellulose powder to a mixture of IOO g of phosphoric acid plus 200 g of urea, and enough water to make fluid. The mixture was boiled for i h at about i i 7 °, cooled, diluted with I 1 of IO % HC1 and washed well with water. Titration in 0.2 M NaC1 showed approx, o.I mequivalent of acid/g of dry powder.
Diet preparation and rabbit growth To ensure uniform distribution of p-fluorophenylalanine throughout the diet, commercial rabbit food pellets were crumbled by dampening with water and kneading. The p-fluorophenylalanine was sprinkled over the crumbled material as a 3 % solution (pH 8) and the mass was again thoroughly kneaded and dried in air over an oven. The addition of 0.5 % p-fluorophenylalanine to the diet of I-lb rabbits caused complete growth inhibition, while an 0.2 ~o level allowed approximately normal growth. In the experiments reported here, animals were fed on a diet containing 0.30 ~o-o.35 ~o p-fluorophenylalanine, a level that allowed approximately half normal growth.
Protein lq7 counting In some early experiments, counting was done with a Packard scintillation Biochim. Biophys. Acta, 54 (I96I) I45-I56
p-FLUOROPHENYLALANINEINCORPORATION
147
counter, using Hyamine (Rohm and Haas) to dissolve the protein in the scintillation medium 21. Most of the results reported were obtained using a gas-fllow counter, and were usually corrected for self-absorption b y plating duplicate samples with added [14c~p-fluorophenylalanine standard. This was of especial importance in counting eluates from D E A E columns since the high and varying salt concentrations of the different fractions caused varying degrees of self-absorption.
Protein separations Aldolase was purified and crystallized b y TAYLOR'S procedure 2. with slight modifications previously described 23, and assayed b y the procedure of JAGANNATHAN et al3 4. Glyceraldehyde 3-phosphate dehydrogenase was purified and crystallized according to VELICK25 and assayed b y D P N H production using conventional procedures as described by BOYER AND SCHULZ25. The muscle protein mixture used for chromatography on D E A E cellulose was a fraction precipitating between 54 % and 82 % saturation of ammonium sulfate at p H 7.4 (measured after 5: I dilution of supernatant); this is the fraction removed from the supernatant of the initial aldolase crystallization prior to crystallization of glyceraldehyde 3-phosphate dehydrogenase. Fractions were dialyzed against starting buffer prior to application to the chromatographic column. Columns used were 2 × 30 cm and were packed under IO lbs./in ~ of nitrogen pressure. Serum proteins were obtained b y withdrawing blood from the abdominal aorta of an ether anaesthetized animal with a heparinized syringe. The sample was then dialyzed, with stirring, against a liter of 0. 9 % NaC1 containing o.oi M phosphate buffer, p H 7-5, and lO -4 M E D T A for about 15 h at 3 °, then twice against ion-low water for 3 and I h. The solution was centrifuged and the clear supernatant lyophilized and stored at - - 2 0 ° until used. The dry material was completely soluble on subsequent addition of cold buffer. Column electrophoreses were performed in columns 3.2 :< 14o cm as described b y FLODIN AND KUPKE 2°. Protein concentrations were measured as the absorbancy of the solution at 280 m/z in a Beckman DU Spectrophotometer, unless otherwise noted. Biuret protein determinations were carried out as described b y LAYNE27, using freshly prepared reagents and running standard curves with the same reagents on the same day.
Hydrolysis and paper chromatography Radioactive recrystallized aldolase was hydrolyzed in 6 N HC1 for 12 h at IiO ° in sealed tubes. Tubes were evacuated with a water p u m p prior to sealing to avoid humin formation; otherwise even added p-fluorophenylalanine was not observed on chromatograms. Fractionation b y ascending chromatography was made using the system described by BAKER et al. ~. The hydrolysate was applied in a line I cm from the longer edge of a 23 × 55 cm sheet of W h a t m a n No. I filter paper. Separate spots of nonradioactive p-fluorophenylalanine were applied at the ends of the line. The paper was then rolled into a cylinder, the edges stapled together, and the cylinder put into a beaker of slightly larger diameter containing approx. 3-4 m m of solvent. A greased lid was put in place and the chromatograph developed at 4 ° b y ascending solvent flow over a period of about 24 h. The paper was then removed from the beaker and Biochim. Biophys. Ac/a, 54 (1961) 145-156
148
E . W . WESTHEAD, P. D. BOYER
the stapled edges cut off, leaving sufficient margin for ninhydrin development of the separate p-fluorophenylalanine spots. The distance migrated b y these spots was then measured, and a strip 2 cm wide cut from the main body of the sheet at the same distance from the origin. I t was evident that this should not include any significant amount of natural amino acid. This strip was then eluted and the eluate plated and counted. Fractionation of hydrolysates by descending paper chromatography was made without the addition of carrier fluorophenylalanine. The same solvent system was used, but hydrolysates were applied as spots, and standard amino acids were run in parallel.
Carboxypeptidase digestion o/aldolase 35 mg of fluorophenylalanine containing aldolase from rabbit No. 3 were incubated with I / z g carboxypeptidase in a volume of 2 ml. After a 4o-min digestion at room temperature, the solution was made lO -3 M in added fluorophenylalanine, and then heated to IOO° for 4 min. After repeated centrifugations and washings of the precipitated protein, the supernatant and washings were put through a "millipore" filter and plated for counting. RESULTS
Growth o/ rabbits /ed p-l~uorophenylalanine The growth of rabbits used for the experiments reported herein is summarized in Table I. The data indicate that addition of 3-3.5 g of p-fluorophenylalanine/ioo g of dry ration is necessary for 50 % growth inhibition. TABLE I GROWTH OF RABBITS FED RADIOACTIVE p-FLUOROPHENYLALAlqINE Rabbit No.
I 2 3
p-FluorophenyiaIanine per 20o g ration (g)
3-5 3-5 to 3.o* 3.o to 3.5*
Weeks fed
2.5 4 7
Initial weight
Final weight
532 368 3 °o
848 574 857
* Adjusted in direction noted during course of e x p e r i m e n t in order to m a i n t a i n approx. 1/2 n o r m a l g r o w t h rate.
Demonstration o~ incorporation of intact p-fluorophenylalanine into proteins The finding of measurable radioactivity in protein fractions from rabbits fed the radioactive p-fluorophenylalanine indicated incorporation of the fluoro amino acid. The findings of ARMSTRONG AND LEWIS*s that some free fluoride is excreted in the urine of rats fed fluorophenylalanine made it necessary to identify the occurrence of 14C in the proteins with the presence of p-fluorophenylalanine as such. For this purpose, a well-defined pure protein, aldolase, was isolated and the nature of the radioactivity present determined. A 9o-mg sample of alkali-denatured, recrystallized aldolase from rabbit No. i was hydrolyzed in 6 N HC1 and after addition of carrier p-fluorophenylalanine to a level of 3 %, treated as described by B i o c h i m . B i o p h y s . A c t a , 54 (1961) 145-156
p-FLUOROPHENYLALANINE
149
INCORPORATION
BAKER et al. ~ to remove humin and to concentrate the aromatic amino acids. The concentrated aromatic amino acid fraction was used for ascending chromatography as described in the experimental section. The strip corresponding to authentic pfluorophenylalanine, run in parallel, was eluted. The observed 14C activity in this sample (165 counts/min above background) was 87 % of the radioactivity of the starting hydrolysate. This probably means that all of the radioactivity in the hydrolysate is due to p-fluorophenylalanine, since "tailing" of the fluorophenylalanine spot plus non-quantitative elutions could easily account for the 13 % difference. There remains, however, the possibility that a small fraction of the 14C activity does appear in other amino acids. The degree of incorporation of p-fluorophenylalanine into the proteins of rabbit No. 3 as determined by specific radioactivity was high enough to allow visualization on a chromatogram without added carrier. For this purpose, a sample of recrystallized glyceraldehyde 3-phosphate dehydrogenase was hydrolyzed and a 2o-/21 aliquot equivalent to about o.2 mg hydrolyzed protein was chromatographed. A faint but distinct spot of ninhydrin-stained material was visible at the position corresponding to p-fluorophenylalanine, well separated from other spots.
Extent of p-fluorophenylalanine incorporation Table II shows the extent of incorporation of p-fluorophenylalanine into crystalline aldolase and glyceraldehyde dehydrogenase in the three rabbits used. Rabbit No. i, which was fed for a shorter period and showed less fractional increase in weight, showed considerably less incorporation than rabbits 2 and 3- Considerable replacement of phenylalanine was shown by rabbit No. 3 ; 1/4 of the p-fluorophenylalanine residues of the aldolase were replaced by p-fluorophenylalanine. The rabbit chow contained TABLE
II
p-FLUOROPHENYLALANINE CONTENT AND CATALYTIC ACTIVITY OF RABBIT MUSCLE ALDOLASE AND GLYCERALDEHYDE B-PHOSPHATE DEHYDROGENASE
Specific 1'C activity of administered p-fluorophenylalanine (l,C/mmole)
Enzyme source
Enzyme
Radioactivity Fluorophenylalanine in enzyme incorporation* (counts/rain/rag) (moles/mole protein)
Phenylalanine replaced** (%)
Rabbit
i
25.8
Aldolase Dehydrogenase
26 26
o. 17 o.12
Rabbit
2
2. 4
Aldolase Dehydrogenase
28 26
3.9 2.6
I5 8
t(~.5 4°
Rabbit
3
z. 4
Aldolase Dehydrogenase
27 3°
6.3 4,9
24 1.5
r7 3 ~)
Normal Control
Aldolase Dehydrogenase
o.65 0.36
Enzyme specific activity
13.o * * * 37
i7 37 42
* C a l c u l a t e d a s s u m i n g t h a t o n l y t h e r a d i o a c t i v i t y f r o m L - ; 3 - 1 4 C ] p - f l u o r o p h e n y l a l a n i n e is i n corporated. ** A l d o l a s e c o n t a i n s 26 a n d t h e d e h y d r o g e n a s e 33 m o l e s of p h e n y l a l a n i n e / m o l e p r o t e i n a2. *** A t t h e t i m e of t h i s a s s a y 13 w a s t h e u s u a l s p e c i f i c a c t i v i t y o b t a i n e d w i t h o u r b e s t a l d o l a s e p r e p a r a t i o n s . H i g h e r a c t i v i t i e s l a t e r r e p o r t e d a r e t h e r e s u l t of p u r i f i c a t i o n o f c o m m e r c i a l f r u c t o s e diphosphate to remove inhibitory inorganic phosphate.
B i o c h i m . B i o p h y s . A c t a , 54 (1961) 1 4 5 - 1 5 6 .
150
E . W . WESTHEAD, P. D. BOYER
18 % protein (manufacturer's assay) ; assuming that 3 % of this is phenylalanine, then the ratio of phenylalanine to fluorophenylalanine in the diet is of the order of 2:1. The high ratio of fluorophenylalanine to normal phenylalanine in the proteins then is strong indication that there is little or no selectivity between phenylalanine and fluorophenylalanine by the protein synthesizing systems of the rabbit. Proteins of blood plasma, soluble proteins of liver, and proteins of a muscle fraction were examined for radioactivity after separation by chromatographic and electrophoretic procedures to see if the incorporation of p-fluorophenylalanine into tissue proteins was a general phenomenon. Results are shown in Figs. I through 3, and demonstrate the generalized incorporation of p-fluorophenylalanine. The amounts incorporated per milligram of protein are roughly in the range observed with the isolated glyceraldehyde 3-phosphate dehydrogenase and aldolase.
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Fig. I. E l u t i o n p a t t e r n f r o m c o l u m n electrophoresis of blood s e r u m f r o m a p - f l u o r o p h e n y l a l a n i n e f e d rabbit. S e r u m f r o m r a b b i t No. 3 w a s s u b j e c t e d to c o l u m n electrophoresis in o.o 3 ionic s t r e n g t h p o t a s s i u m p h o s p h a t e buffer, p H 7.3 for 24 h a t 42 m A , t h e n e l u t e d w i t h t h e s a m e buffer solution. T h e vertical b a r s indicate t h e 14C a c t i v i t y in c o u n t s / m i n / m l of solution of u n i t a b s o r b a n c y a t 28o m/~ (right h a n d ordinate); t h e solid line gives t h e A 380 of t h e effluent.
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Fig. 2. E l u t i o n p a t t e r n f r o m c h r o m a t o g r a p h y of m u s c l e p r o t e i n s f r o m a p - f l u o r o p h e n y l a l a n i n e fed r a b b i t . A n e x t r a c t f r o m r a b b i t No. i p r e p a r e d as described in t h e t e x t w a s a d s o r b e d o n a D E A E cellulose c o l u m n , t h e n e l u t e d w i t h 35 ° m l of o.005 ionic s t r e n g t h T r i s - H C 1 buffer, p H 7.7, followed b y a " l i n e a r " g r a d i e n t u s i n g 2oo m l each of s t a r t i n g buffer a n d o.Io ionic s t r e n g t h Tris, p H 7.7. T h e s y m b o l s L D H a n d P K refer to location of p e a k s c o n t a i n i n g h i g h activities of lactic d e h y d r o g e n a s e a n d p y r u v a t e kinase. Vertical b a r s i n d i c a t e relative 14C activities as in Fig. i.
Biochim. Biophys. Acta, 54 (1961) 145-156
p-FLUOROPHENYLALANINEINCORPORATION
151
Evidence/or replacement o[ phenylalanine Structural relationships indicate that p-fluorophenylalanine would be expected to replace phenylalanine or tyrosine, and bacterial data 5,18,19 strongly suggest phenylalanine replacement. The relative extents of incorporation of p-fluorophenylalanine into aldolase and glyceraldehyde 3-phosphate dehydrogenase are in approximate proportion to their tyrosine contents. However, two lines of evidence demonstrate that p-fluorophenylalanine does not readily replace tyrosine in rabbit proteins and thus likely replaces phenylalanine predominately or entirely. The difference in extent of replacement of phenylalanine in aldolase and glyceraldehyde 3-phosphate dehydrogenase as noted in DISCUSSION,probably reflects different turnover rates. Carboxypeptidase is known to remove only three COOH terminal tyrosine and two alanine residues from aldolase 23. Replacement of these tyrosines by fluorophenyl-
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Fig. 3. Elution pattern from chromatography of liver proteins from a p-fluorophenylalanine-fed rabbit. Frozen liver was homogenized in o.oi ionic strength Tris buffer, pH 7.4, the mixture centrifuged at about 120oo × g for 15-2o min, the supernatant solution dialyzed 12 h against ioo volumes of the buffer, and the soluble extract used for chromatography on DEAE cellulose. The column was eluted using a gradient from o.oo5 ionic strength Tris HC1 buffer, pH 7.3 to o.o5 ionic strength Tris-HC1 buffer, pH 7.3; 200 ml of each buffer. Vertical bars indicate relative 14C activities as in Fig. I. alanine should result in the appearance of detectable radioactivity in the supernatant solution from precipitation of carboxypeptidase digested aldolase. This was tested experimentally by digestion of 35 mg of three times recrystallized aldolase from rabbit No. 3- Random substitution for tyrosine would have resulted in a total of 66 counts/min over background in the supernatant. Instead, 7 counts/min over background was found, indicating that little, if any, of the fluorophenylalanine was in place of the terminal tyrosines. The 7 counts/min could have been due to a small amount of filterable protein, or to fluorophenylalanine derived from denatured aldolase by carboxypeptidase action. Additional demonstration of phenylalanine replacement comes from a comparison of the biuret color with the 28o-m/, absorbancy for the two crystalline proteins. The biuret color is due to peptide-copper chelates and thus would be insensitive to substitution of p-fluorophenylalanine for tyrosine. Replacement of tyrosine would lower the ratio of 28o-m/, absorption to biuret color. Fig. 4 shows the relation between 28o-m/~ absorbancy and biuret color at 54 ° m/x for aldolase and
Biochim. Biophys. Acta, 54 (I96I) I45-I56
152
E. W. WESTHEAD, P. D. BOYER
glyceraldehyde dehydrogenase, together with experimental points for the fluorophenylalanine containing enzymes. The precision of the method is such that replacement to the extent of more than 3 ~0 would lead to significant deviation of the experimental points from the standard curves, whereas on the basis of radioactivity nearly 20 ~o of the tyrosine could have been replaced by the analog. '
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0.2
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Fig. 4. Relative reaction with biuret and absorbancy at 28o m# of normal and of p-fluorophenylalanine-containing aldolase (Fig. 4a) and glyceraldehyde 3-phosphate dehydrogenase (Fig. 4b). The values obtained for the A280 as measured directly are given on the abscissa, and the values obtained for the A540 in the biuret determination for the same final protein concentration are given on the ordinate. © - - © , Normal aldolase; O - - O , p-fluorophenylalanine-containing aldolase; [ ] - - •, normal glyceraldehyde 3-phosphate dehydrogenase; • - - I , p-fluorophenylalaninecontaining glyceraldehyde 3-phosphate dehydrogenase.
Effects o/p-fluorophenylalanine incorporation on protein properties Both aldolase and glyceraldehyde dehydrogenase containing p-fluorophenylalanine crystallized under the same conditions used for the crystallization of the normal enzymes. Results of measurement of enzymic activity are shown in the last line of Table I. There is no significant difference between the activities of the normal and radioactive enzymes. Chromatographic and electrophoretic separations were made as further tests of possible differences in properties of normal and p-fluorophenylalanine-containing enzymes. In addition to differences from normal enzymes, individual molecules of the aldolase and dehydrogenase preparations from experimental animals might reasonably be expected to contain different amounts of p-fluorophenylalanine. Such differences could result because following ingestion of the ration, the added p-fluorophenylalanine would likely reach the tissues before the bulk of the phenylalanine, and the rates of metabolism of phenylalanine and p-fluorophenylalanine may differ. Thus the protein synthesizing systems were likely presented with variable p-fluorophenylalanine/phenylalanine ratios through a given day. Small differences in properties reflecting differences in extent of incorporation might be detectable by the techniques used. Fig. 5 shows the result of chromatography of aldolase from rabbit No. 3 on a phosphorylated cellulose column. The protein used was the first crop of crystals from muscle extract, with specific activity within IO °/o of that shown by our purest aldolase preparations. The specific activity and specific radioactivity ratios Biochim. Biophys. +4eta, 54 (1961) 145-156
153
p-FLUOROPHENYLALANINE INCORPORATION
indicate no detectable heterogeneity with respect to fluorophenylalanine content. Chromatography of normal aldolase on phosphorylated cellulose has invariably given patterns similar to that in Fig. 5, despite the fact that chromatography on another cation exchanger, carboxymethyl cellulose yields a single peak. Rechromatography of the main peak from the elution of normal aldolase gave a single peak, indicating
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l"ig. 5. Elution p a t t e r n from c h r o m a t o g r a p h y of fluorophenylalanine-containing aldolase on p h o s p h o r y l a t e d cellulose. Crystalline aldolase from r a b b i t No. 3 was dissolved in o.o I ionic s t r e n g t h Tris HC1, p H 7.6, and adsorbed on a p h o s p h o r y l a t e d cellulose column, t h e n eluted w i t h a " l i n e a r " gradient from 75 ml of the same buffer to 75 ml of this buffer made 0. 5 ~'~ in NaC1. Vertical b a r s indicate relative 14C activities as in Fig. i.
that this heterogeneity is not an artifact of chromatography. A more complete discussion of the chromatographic behavior of normal aldolase, together with evidence for the catalytic homogeneity of the enzyme, will be given in another paper now in preparation. Fig. 6 shows the elution pattern of a sample of aldolase from rabbit, once recrystallized, from a column electrophoresis. Again, specific activity and specific radio&
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Fig. 6. E l u t i o n p a t t e r n from c o l u m n electrophoresis of fluorophenylalanine-containing aldolase. Crystalline aldolase from r a b b i t No. 3 was subjected to c o l u m n electrophoresis for 3 ° h, 35 ° V, in Tris-HC1 buffer, o.o 5 ionic strength, p H 7.8, t h e n eluted w i t h the same buffer. The broken line indicates specific enzymic activity; the triangles indicate relative 14C c o n t e n t of fractions as c o u n t s / m i n / m l of solution of u n i t a b s o r b a n c y at 280 m/~.
Biochim. Biophys. Acta, 54 (1961) 145 156
154
E. W. WESTHEAD, P, D. BOYER
activity measurements indicate no heterogeneity based on p-fluorophenylalanine content or activity. In order to determine whether or not the presence of p-fluorophenylalanine in the molecule affected the physical stability of the enzyme, a solution of four times recrystallized glyceraldehyde 3-phosphate dehydrogenase was heated until appreciable protein precipitated. The precipitate was then centrifuged down, the protein concentration of the supernatant measured, and the "C activity determined. This was repeated several times. As indicated in Fig. 7, the "C activity of the supernatant remained proportional to the protein content, showing that there was no preferential precipitation of protein with either high or low p-fluorophenylalanine content. As a further check, a sample of several times recrystallized glyceraldehyde 3-phosphate dehydrogenase from a normal rabbit was mixed with a sample of p-fluorophenylalanine containing enzyme in approximately equal amounts. Stepwise heat denaturation again failed to show any difference in stability between the normal and the p-fluorophenylalanine-protein (Fig. 7).
0
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i .o 40
Y I
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I I .~oo ABSORBANCY
.too
290
I .4oo m~
I .ooo
Fig. 7- Stepwise heat denaturation of p-fluorophenylalanine-containing glyceraldehyde dehydrogenase. / X - - - A , Mixed sample of p-fluorophenylalanine enzyme plus normal enzyme; © - - - O , fluorophenylalanine enzyme alone. Samples were heated as described in the text, the insoluble protein removed, and the A 290 and radioactivity remaining in solution were determined. Measurements were made at A290 to avoid the absorbancy of the bound diphosphopyridine nucleotide. DISCUSSrON
The results show clearly that p-fluorophenylalanine is readily incorporated into a variety of proteins by the rabbit. The incorporation appears to occur chiefly or wholly by replacement of phenylalanine residues. The relatively high degree of incorporation found demonstrates the occurrence of little selectivity of phenylalanine over p-fluorophenylalanine by the protein synthesizing systems. A lack of prominent interference of p-fluorophenylalanine incorporation in the function of various proteins is indicated by the survival of the animal even though extensive incorporation occurs. The findings with the isolated aldolase and glyceraldehyde 3-phosphate dehydrogenase demonstrate no reduction of catalytic activity although, with aldolase, as much as 25 % of the phenylalanine residues have been replaced. Similarly, no differences in other properties of the proteins was found. Aldolase and glyceraldehyde 3-phosphate dehydrogenase fractionated and crystallized Biochim. Biophys. Acta, 54 (1961) I45-I56
p-FLUOROPHENPLALANINE INCORPORATION
155
in a normal manner, and the phenylalanine replacement did not alter the chromatographic behavior of the aldolase or the heat sensitivity of the dehydrogenase. Also, the electrophoretic pattern of blood plasma proteins was not appreciably modified. That decreased enzyme activity may accompany p-fluorophenylalanine incorporation is shown, however, b y the decrease in the activity of bacterial amylase e and penicillinase 7. The studies of RICHMOND with penicillinase 7 are particularly convincing; a high percentage of phenylalanine replacement was obtained, accompanied by a large reduction in enzyme activity and change in immunological properties. The apparently inert phenyl side chain can thus play a significant role in the tertiary structure of some proteins. The attempt to locate one or more mammalian enzymes showing critical sensitivity toward an incorporated analog may prove difficult. Perhaps a more hopeful approach, and one that is likely to be more fruitful as additional information is available about the composition of "active sites" and structural features of enzymes, is the search for specific analogs to replace amino acids known to be critical for the activity of particular enzymes. The present work was undertaken partly with this intention. A report that fluorophenylalanine replaced tyrosine in part 3°, made it appear likely that aldolase activity would be modified if fluorophenylalanine could be incorporated, since it was known that tyrosine is intimately involved with aldolase activity 23. The report was later withdrawn when the observation was found to be the result of an artifact of hydrolysis 5. The demonstration of widespread incorporation of p-fluorophenylalanine into blood, liver, and muscle proteins in about the same amounts indicates a lack of selectivity of the various protein-synthesizing systems for the natural amino acids. Some differences in extent of incorporation are to be expected because of the differences in rates of turnover of mammalian proteins. This very likely accounts for the greater degree of p-fluorophenylalanine substitution in aldolase than in glyceraldehyde 3-phosphate dehydrogenase. SIMPSON AND VELICK have shown that aldolase turns over nearly twice as fast as the dehydrogenase 29. Our data by no means proves that selectivity does not exist in some instances, and, indeed, it is possible that selectivity may be manifest when analog incorporation would result in interference in essential activity. The possibility of selectivity is indicated by the data of RABINOVITZ AND MCGRATH showing that both o-fluorophenylalanine and a-amino-fl-chlorobutyrate inhibit protein synthesis in reticulocytes but not in Ehrlich ascites cells~1. Studies with bacteria, which can be grown to many times their initial mass, are more likely to provide evidence about relative selectivity for different analogs of systems synthesizing various proteins. COWIE et al., in a series of papers, have demonstrated the relative uniformity of incorporation of a variety of analog into proteins separated on DEAE cellulose columns 4. However, despite their clear evidence that gross differences do not exist in the degrees of incorporation into a large number of protein fractions, their results do not rule out the possibility that sharp differences in degree of incorporation exist in minor components of the protein mixture. The only method of providing direct evidence about selectivity is through isolating and purifying various individual proteins as has been done by STEINBERGet al. 14, YOSHIDA AND YAMASAKI1°, and in the present work. This small amount of evidence indicates that for the 5 proteins isolated, incorporation into the individual proteins approximately parallels the incorporation into other proteins. YOSHIDAAND Y.~MASAKI Biochirn. Biophys. Acta, 54 (1961) 145-156
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E . W . WESTHEAD, P. D. BOYER
have further been able to show that the degree of replacement of methionine by ethioDine is the same in each of a number of tryptic fragments as in the complete protein. Although our studies did not reveal any modification of enzyme activity as a result of p-fluorophenylalanine incorporation, the possibility remains open that the toxicity of p-fluorophenylalanine to mammals results largely or in part from an altered biological activity of certain proteins. Another possibility is that although an analog may perform satisfactorily in a protein, it may inhibit or produce inhibitory side products in other metabolic processes to which it is subjected. As pointed out by VAUGHAN A N D STEINBERG18, a number of analogs that have been incorporated into proteins are known to undergo other metabolic transformations and toxicity resulting from any effects on protein structure or function are often secondary to effects produced in other ways. ACKNOWLEDGEMENTS
This investigation was supported in part by U.S. Public Health Service Research Grants RG-493o and A-344I, U.S. Atomic Energy Commission Contract AT (II-I)341 and the Hill Family Foundation. REFERENCES 1 A. B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 27 (1958) 33 o. 2 G. BRAWERMAN AND M. YCAS, Arch. Biochem. Biophys., 68 (1957) 112. 3 R. S. BAKER, J. E. JOHNSON AND S. W. FOX, Biochim. Biophys. Acta, 28 (1958) 318. 4 D . B . COWIE, G. N . COHEN, E . T . BOLTON AND H . DE ROBICHON-SZULMAJSTER, Biochim. Biophys. Acta, 34 (1959) 39. 5 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 31 (1959) 378 6 A. YOSHIDA Biochim. Biophys. Acta, 41 (196o) 98. 7 M. H. RICHMOND, Biochem. J., 77 (196o) 112, 121. 8 D. B. COWIE AND G. N. COHEN, Biochim. Biophys. Acta, 26 (1957) 252. 9 D. GROS AND H. TARVER, J. Biol. Chem., 217 (1955) 169. 10 A. YOSHIDA AND M. YAMASAKI, Biochim. Biophys. Acta, 34 (1959) 158. 11 M. H. RICHMOND, BiOchem. J., 73 (1959) 261. 12 A. L. BLACK AND M. KLEIBER, J. Am. Chem. Soc., 77 (1955) 6082. 13 E. W. ~¥'ESTHEAD AND P. D. BOYER, A~$. Chem. Sac. Abstracts, September 1958 , p. 2c. 14 D . STEINBERG, M. VAUGHAN AND F. G. SHERMAN, Biochim. Biophys. Aeta, 4 ° (196o) 225. 15 j . KRUH AND J. RosA, Biochim. Biophys. Acta, 34 (1959) 561. 16 p. F. 1{RUSE, P. B. WHITE, H. A. CARTER AND T. A. McCoY, Cancer Research, 19 (1959) 122. 17 M. RABINOVITZ AND RUTH K. TUVE, Proc. Soc. Exptl. Biol. Med., IOO (1959) 222. 18 M. VAUGHAN AND D. STEINBERG, Advances in Protein Chem., Vol. 14, A c a d e m i c Press, N e w Y ork, 1959, p. 115. 19 G. N. COHEN AND F. GROS, Ann. Rev. Biochem., 29 (196o) 525 . s0 p. FLODIN AND D. V~'. KUPKE, Biochim. Biophys. Acta, 21 (1956) 368. 21 M. VAUGHAN, D. STEINBERG AND J. LOGAN, Science, 126 II957) 446. 23 j . F. TAYLOR, A. A. GREEN AND G. T. CORI, J. Biol. Chem., 173 (1948) 591 . 23 E. R. DRECHSLER, P. D. BOYER AND A. G. KOWALSKY, J. Biol. Chem., 234 (1959) 2627. 24 V. JAGANNATHAN, K. SINGH AND M. DAMODARAN, Biochem. J., 63 (1956) 9425 S. F. VELICK, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, A c a d e m i c P r e s s , N e w York, 1955, Vol. I, p. 4Ol. 26 p. D. BOYER AND A. R. SCHULZ, in R. BENESCH et al., Sulfur in Proteins, A c a d e m i c P r e s s , N e w Yo rk , 1959, p. 199. 27 E. LAYNE, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 3, A c a d e m i c P r e s s , N e w Y o r k , 1955, p. 447. 33 M. D. ARMSTRONG AND J. D. L E w i s , J. Biol. Chem., 19o (1951) 461. 29 M. V. SIMPSON AND S. F. VELICK, J. Biol. Chem., 208 (1954) 61. 30 R. MUNIER AND G. N. COHEN, Bioehim. Biophys. Acta, 21 (1956) 592. 31 M. RABINOVlTZ AND H. MCGRATH, J. Biol. Chem., 234 (1959) 2091. 32 S. F. VELICK AND V.. RONZONI, J. Biol. Chem., 173 (1948) 627.
Biochim. Biophys. Acta, 54 (1961) 145-156
THE INCORPORATION OF p-FLUOROPHENYLALANINE
145
INTO
SOME R A B B I T E N Z Y M E S A N D O T H E R P R O T E I N S E. W. W E S T H E A D * AND P. D. B O Y E R
Department o[ Physiological Chemistry, University of Minnesota, Minneapolis, Minn. (U.S.A.) (Received March 2oth, 1961)
SUMMARY
p-Fluorophenyl-E3-14Clalanine, when fed to rabbits, is incorporated into proteins of muscle, blood, and liver. The incorporation is extensive and takes place to approximately the same extent among proteins fractionated in a variety of ways. The p-fluorophenylalanine does not substitute for tyrosine to any measurable extent and is presumed to replace phenylalanine. Aldolase with a replacement of 25 % and glyceraldehyde 3-phosphate dehydrogenase with a replacement of approx. 16 % of the phenylalanine residues were crystallized in good yields under the same conditions used for the normal enzymes. Enzymic activities of the crystallized p-fluorophenylalanine-containing enzymes were the same as those of normal enzymes. The aldolase was chromatographically homogeneous with respect to p-fluorophenylalanine content and specific activity. Heat denaturation of glyceraldehyde 3-phosphate dehydrogenase showed that there was no detectable difference between the stabilities of the normal enzyme and the p-fiuorophenylalanine-containing enzyme.
INTRODUC~ON
Considerable evidence has accrued that the protein synthesizing mechanisms of bacteria do not show an absolute specificity for the natural amino acids. For example, 7-azatryptophan 1 and tryptazane ~ may substitute for tryptophan; p-fluorophenylalanine 3-7 and fl-2-thienylalanine 5 may substitute for phenylalanine; norleucine 5, selenomethionine s, and ethionine 9,1° may substitute for methionine; and canavanine may substitute for arginine n. In contrast, meager data are available concerning amino acid analog incorporation by mammalian systems. Reports from studies with intact animals appear to be limited to the demonstration of incorporation of norleucine into casein by cows 12 and the preliminary report 13 of the studies given in more detail herein demonstrating incorporation of p-fluorophenylalanine into rabbit proteins. I n vitro studies have shown incorporation of p-fluorophenylalanine into lysozyme and ovalbumin by minced hen oviducO 4 and into hemoglobin by erythrocytes x~, of canavanine into tumor cells in tissue culture 16, and of lysine analogs by rat-bone marrow preparationslL * Present address: D e p a r t m e n t of Biochemistry, D a r t m o u t h Medical School, Hanover, N.H.
(U.S.A.). Biochim. Biophys. Acta, 54 (1961) I 4 5 - I 5 ~
140
E. W. WESTFIEAD, P. D. BOYER
Information from and implications of studies relating to the specificity of protein synthesis have recently been ably reviewed by VAUGI~AN AND STEINBERGis. Recent information on this topic is also included in a review by COHENAND GROS19. A question of considerable importance is to what extent the incorporation of an amino acid analog may modify the molecular and functional properties of a particular protein. Evidence from bacterial studies indicates that in presence of some analogs proteins are synthesized with a modified composition but which are catalytically activO s. Further, crystalline B. subtilis a-amylase containing considerable ethionine retains full catalytic activity 1°. Replacement of about 1/6 of the phenylalanine by p-fluorophenylalanine, however, gave an amylase of the same physical properties but only about 7o % of the normal activity 6, and incorporation into penicillinase resulted in extensive activity lossL The studies reported herein were undertaken to find if p-fluorophenylalanine would be incorporated into proteins of the intact animal, and, if so, what readily detectable changes in properties of some proteins might result. EXPERIMENTAL
Materials and chemicals p-Fluoro-DL-phenylalanine was used as obtained from California Biochemical Corporation, Los Angeles 63, California. p-Fluoro-DL-phenylalanine labeled with 14C in the/~ position with an activity of 2.35 mC/mmole was obtained from the French Atomic Energy Commission. The recorded ultraviolet absorption spectrum of this material was identical with that of the California Biochemical Corporation product. Radioactive p-fluorophenylalanine was diluted with non-labeled material before addition to the diet. DEAE cellulose was purchased from Brown Mills, Keene, New Hampshire and washed with I M NaOH until colorless, before use. Ethanolized cellulose~o for column electrophoresis was purchased from A. B. Munktells, Grycksbo, Sweden. Phosphorylated cellulose used for aldolase chromatography was prepared by adding 35 g of Munktell's cellulose powder to a mixture of IOO g of phosphoric acid plus 200 g of urea, and enough water to make fluid. The mixture was boiled for i h at about i i 7 °, cooled, diluted with I 1 of IO % HC1 and washed well with water. Titration in 0.2 M NaC1 showed approx, o.I mequivalent of acid/g of dry powder.
Diet preparation and rabbit growth To ensure uniform distribution of p-fluorophenylalanine throughout the diet, commercial rabbit food pellets were crumbled by dampening with water and kneading. The p-fluorophenylalanine was sprinkled over the crumbled material as a 3 % solution (pH 8) and the mass was again thoroughly kneaded and dried in air over an oven. The addition of 0.5 % p-fluorophenylalanine to the diet of I-lb rabbits caused complete growth inhibition, while an 0.2 ~o level allowed approximately normal growth. In the experiments reported here, animals were fed on a diet containing 0.30 ~o-o.35 ~o p-fluorophenylalanine, a level that allowed approximately half normal growth.
Protein lq7 counting In some early experiments, counting was done with a Packard scintillation Biochim. Biophys. Acta, 54 (I96I) I45-I56
p-FLUOROPHENYLALANINEINCORPORATION
147
counter, using Hyamine (Rohm and Haas) to dissolve the protein in the scintillation medium 21. Most of the results reported were obtained using a gas-fllow counter, and were usually corrected for self-absorption b y plating duplicate samples with added [14c~p-fluorophenylalanine standard. This was of especial importance in counting eluates from D E A E columns since the high and varying salt concentrations of the different fractions caused varying degrees of self-absorption.
Protein separations Aldolase was purified and crystallized b y TAYLOR'S procedure 2. with slight modifications previously described 23, and assayed b y the procedure of JAGANNATHAN et al3 4. Glyceraldehyde 3-phosphate dehydrogenase was purified and crystallized according to VELICK25 and assayed b y D P N H production using conventional procedures as described by BOYER AND SCHULZ25. The muscle protein mixture used for chromatography on D E A E cellulose was a fraction precipitating between 54 % and 82 % saturation of ammonium sulfate at p H 7.4 (measured after 5: I dilution of supernatant); this is the fraction removed from the supernatant of the initial aldolase crystallization prior to crystallization of glyceraldehyde 3-phosphate dehydrogenase. Fractions were dialyzed against starting buffer prior to application to the chromatographic column. Columns used were 2 × 30 cm and were packed under IO lbs./in ~ of nitrogen pressure. Serum proteins were obtained b y withdrawing blood from the abdominal aorta of an ether anaesthetized animal with a heparinized syringe. The sample was then dialyzed, with stirring, against a liter of 0. 9 % NaC1 containing o.oi M phosphate buffer, p H 7-5, and lO -4 M E D T A for about 15 h at 3 °, then twice against ion-low water for 3 and I h. The solution was centrifuged and the clear supernatant lyophilized and stored at - - 2 0 ° until used. The dry material was completely soluble on subsequent addition of cold buffer. Column electrophoreses were performed in columns 3.2 :< 14o cm as described b y FLODIN AND KUPKE 2°. Protein concentrations were measured as the absorbancy of the solution at 280 m/z in a Beckman DU Spectrophotometer, unless otherwise noted. Biuret protein determinations were carried out as described b y LAYNE27, using freshly prepared reagents and running standard curves with the same reagents on the same day.
Hydrolysis and paper chromatography Radioactive recrystallized aldolase was hydrolyzed in 6 N HC1 for 12 h at IiO ° in sealed tubes. Tubes were evacuated with a water p u m p prior to sealing to avoid humin formation; otherwise even added p-fluorophenylalanine was not observed on chromatograms. Fractionation b y ascending chromatography was made using the system described by BAKER et al. ~. The hydrolysate was applied in a line I cm from the longer edge of a 23 × 55 cm sheet of W h a t m a n No. I filter paper. Separate spots of nonradioactive p-fluorophenylalanine were applied at the ends of the line. The paper was then rolled into a cylinder, the edges stapled together, and the cylinder put into a beaker of slightly larger diameter containing approx. 3-4 m m of solvent. A greased lid was put in place and the chromatograph developed at 4 ° b y ascending solvent flow over a period of about 24 h. The paper was then removed from the beaker and Biochim. Biophys. Ac/a, 54 (1961) 145-156
148
E . W . WESTHEAD, P. D. BOYER
the stapled edges cut off, leaving sufficient margin for ninhydrin development of the separate p-fluorophenylalanine spots. The distance migrated b y these spots was then measured, and a strip 2 cm wide cut from the main body of the sheet at the same distance from the origin. I t was evident that this should not include any significant amount of natural amino acid. This strip was then eluted and the eluate plated and counted. Fractionation of hydrolysates by descending paper chromatography was made without the addition of carrier fluorophenylalanine. The same solvent system was used, but hydrolysates were applied as spots, and standard amino acids were run in parallel.
Carboxypeptidase digestion o/aldolase 35 mg of fluorophenylalanine containing aldolase from rabbit No. 3 were incubated with I / z g carboxypeptidase in a volume of 2 ml. After a 4o-min digestion at room temperature, the solution was made lO -3 M in added fluorophenylalanine, and then heated to IOO° for 4 min. After repeated centrifugations and washings of the precipitated protein, the supernatant and washings were put through a "millipore" filter and plated for counting. RESULTS
Growth o/ rabbits /ed p-l~uorophenylalanine The growth of rabbits used for the experiments reported herein is summarized in Table I. The data indicate that addition of 3-3.5 g of p-fluorophenylalanine/ioo g of dry ration is necessary for 50 % growth inhibition. TABLE I GROWTH OF RABBITS FED RADIOACTIVE p-FLUOROPHENYLALAlqINE Rabbit No.
I 2 3
p-FluorophenyiaIanine per 20o g ration (g)
3-5 3-5 to 3.o* 3.o to 3.5*
Weeks fed
2.5 4 7
Initial weight
Final weight
532 368 3 °o
848 574 857
* Adjusted in direction noted during course of e x p e r i m e n t in order to m a i n t a i n approx. 1/2 n o r m a l g r o w t h rate.
Demonstration o~ incorporation of intact p-fluorophenylalanine into proteins The finding of measurable radioactivity in protein fractions from rabbits fed the radioactive p-fluorophenylalanine indicated incorporation of the fluoro amino acid. The findings of ARMSTRONG AND LEWIS*s that some free fluoride is excreted in the urine of rats fed fluorophenylalanine made it necessary to identify the occurrence of 14C in the proteins with the presence of p-fluorophenylalanine as such. For this purpose, a well-defined pure protein, aldolase, was isolated and the nature of the radioactivity present determined. A 9o-mg sample of alkali-denatured, recrystallized aldolase from rabbit No. i was hydrolyzed in 6 N HC1 and after addition of carrier p-fluorophenylalanine to a level of 3 %, treated as described by B i o c h i m . B i o p h y s . A c t a , 54 (1961) 145-156
p-FLUOROPHENYLALANINE
149
INCORPORATION
BAKER et al. ~ to remove humin and to concentrate the aromatic amino acids. The concentrated aromatic amino acid fraction was used for ascending chromatography as described in the experimental section. The strip corresponding to authentic pfluorophenylalanine, run in parallel, was eluted. The observed 14C activity in this sample (165 counts/min above background) was 87 % of the radioactivity of the starting hydrolysate. This probably means that all of the radioactivity in the hydrolysate is due to p-fluorophenylalanine, since "tailing" of the fluorophenylalanine spot plus non-quantitative elutions could easily account for the 13 % difference. There remains, however, the possibility that a small fraction of the 14C activity does appear in other amino acids. The degree of incorporation of p-fluorophenylalanine into the proteins of rabbit No. 3 as determined by specific radioactivity was high enough to allow visualization on a chromatogram without added carrier. For this purpose, a sample of recrystallized glyceraldehyde 3-phosphate dehydrogenase was hydrolyzed and a 2o-/21 aliquot equivalent to about o.2 mg hydrolyzed protein was chromatographed. A faint but distinct spot of ninhydrin-stained material was visible at the position corresponding to p-fluorophenylalanine, well separated from other spots.
Extent of p-fluorophenylalanine incorporation Table II shows the extent of incorporation of p-fluorophenylalanine into crystalline aldolase and glyceraldehyde dehydrogenase in the three rabbits used. Rabbit No. i, which was fed for a shorter period and showed less fractional increase in weight, showed considerably less incorporation than rabbits 2 and 3- Considerable replacement of phenylalanine was shown by rabbit No. 3 ; 1/4 of the p-fluorophenylalanine residues of the aldolase were replaced by p-fluorophenylalanine. The rabbit chow contained TABLE
II
p-FLUOROPHENYLALANINE CONTENT AND CATALYTIC ACTIVITY OF RABBIT MUSCLE ALDOLASE AND GLYCERALDEHYDE B-PHOSPHATE DEHYDROGENASE
Specific 1'C activity of administered p-fluorophenylalanine (l,C/mmole)
Enzyme source
Enzyme
Radioactivity Fluorophenylalanine in enzyme incorporation* (counts/rain/rag) (moles/mole protein)
Phenylalanine replaced** (%)
Rabbit
i
25.8
Aldolase Dehydrogenase
26 26
o. 17 o.12
Rabbit
2
2. 4
Aldolase Dehydrogenase
28 26
3.9 2.6
I5 8
t(~.5 4°
Rabbit
3
z. 4
Aldolase Dehydrogenase
27 3°
6.3 4,9
24 1.5
r7 3 ~)
Normal Control
Aldolase Dehydrogenase
o.65 0.36
Enzyme specific activity
13.o * * * 37
i7 37 42
* C a l c u l a t e d a s s u m i n g t h a t o n l y t h e r a d i o a c t i v i t y f r o m L - ; 3 - 1 4 C ] p - f l u o r o p h e n y l a l a n i n e is i n corporated. ** A l d o l a s e c o n t a i n s 26 a n d t h e d e h y d r o g e n a s e 33 m o l e s of p h e n y l a l a n i n e / m o l e p r o t e i n a2. *** A t t h e t i m e of t h i s a s s a y 13 w a s t h e u s u a l s p e c i f i c a c t i v i t y o b t a i n e d w i t h o u r b e s t a l d o l a s e p r e p a r a t i o n s . H i g h e r a c t i v i t i e s l a t e r r e p o r t e d a r e t h e r e s u l t of p u r i f i c a t i o n o f c o m m e r c i a l f r u c t o s e diphosphate to remove inhibitory inorganic phosphate.
B i o c h i m . B i o p h y s . A c t a , 54 (1961) 1 4 5 - 1 5 6 .
150
E . W . WESTHEAD, P. D. BOYER
18 % protein (manufacturer's assay) ; assuming that 3 % of this is phenylalanine, then the ratio of phenylalanine to fluorophenylalanine in the diet is of the order of 2:1. The high ratio of fluorophenylalanine to normal phenylalanine in the proteins then is strong indication that there is little or no selectivity between phenylalanine and fluorophenylalanine by the protein synthesizing systems of the rabbit. Proteins of blood plasma, soluble proteins of liver, and proteins of a muscle fraction were examined for radioactivity after separation by chromatographic and electrophoretic procedures to see if the incorporation of p-fluorophenylalanine into tissue proteins was a general phenomenon. Results are shown in Figs. I through 3, and demonstrate the generalized incorporation of p-fluorophenylalanine. The amounts incorporated per milligram of protein are roughly in the range observed with the isolated glyceraldehyde 3-phosphate dehydrogenase and aldolase.
g c z -I o
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5o
-~ %.
>-
~=
so ~
~t,o
Y
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|oo
BOO
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EFFLUENT VOLUME,ML
Fig. I. E l u t i o n p a t t e r n f r o m c o l u m n electrophoresis of blood s e r u m f r o m a p - f l u o r o p h e n y l a l a n i n e f e d rabbit. S e r u m f r o m r a b b i t No. 3 w a s s u b j e c t e d to c o l u m n electrophoresis in o.o 3 ionic s t r e n g t h p o t a s s i u m p h o s p h a t e buffer, p H 7.3 for 24 h a t 42 m A , t h e n e l u t e d w i t h t h e s a m e buffer solution. T h e vertical b a r s indicate t h e 14C a c t i v i t y in c o u n t s / m i n / m l of solution of u n i t a b s o r b a n c y a t 28o m/~ (right h a n d ordinate); t h e solid line gives t h e A 380 of t h e effluent.
I.t
L DH
o
EFFLUENT VOLUME, ML
Fig. 2. E l u t i o n p a t t e r n f r o m c h r o m a t o g r a p h y of m u s c l e p r o t e i n s f r o m a p - f l u o r o p h e n y l a l a n i n e fed r a b b i t . A n e x t r a c t f r o m r a b b i t No. i p r e p a r e d as described in t h e t e x t w a s a d s o r b e d o n a D E A E cellulose c o l u m n , t h e n e l u t e d w i t h 35 ° m l of o.005 ionic s t r e n g t h T r i s - H C 1 buffer, p H 7.7, followed b y a " l i n e a r " g r a d i e n t u s i n g 2oo m l each of s t a r t i n g buffer a n d o.Io ionic s t r e n g t h Tris, p H 7.7. T h e s y m b o l s L D H a n d P K refer to location of p e a k s c o n t a i n i n g h i g h activities of lactic d e h y d r o g e n a s e a n d p y r u v a t e kinase. Vertical b a r s i n d i c a t e relative 14C activities as in Fig. i.
Biochim. Biophys. Acta, 54 (1961) 145-156
p-FLUOROPHENYLALANINEINCORPORATION
151
Evidence/or replacement o[ phenylalanine Structural relationships indicate that p-fluorophenylalanine would be expected to replace phenylalanine or tyrosine, and bacterial data 5,18,19 strongly suggest phenylalanine replacement. The relative extents of incorporation of p-fluorophenylalanine into aldolase and glyceraldehyde 3-phosphate dehydrogenase are in approximate proportion to their tyrosine contents. However, two lines of evidence demonstrate that p-fluorophenylalanine does not readily replace tyrosine in rabbit proteins and thus likely replaces phenylalanine predominately or entirely. The difference in extent of replacement of phenylalanine in aldolase and glyceraldehyde 3-phosphate dehydrogenase as noted in DISCUSSION,probably reflects different turnover rates. Carboxypeptidase is known to remove only three COOH terminal tyrosine and two alanine residues from aldolase 23. Replacement of these tyrosines by fluorophenyl-
::1.1.4 -
D Z
E 01,2 cJ
•° ~o W 3oZ
o
o~
-4 10
I 0
i I00
~00
tOO
EFFLUENT
VOLUME,
ML
Fig. 3. Elution pattern from chromatography of liver proteins from a p-fluorophenylalanine-fed rabbit. Frozen liver was homogenized in o.oi ionic strength Tris buffer, pH 7.4, the mixture centrifuged at about 120oo × g for 15-2o min, the supernatant solution dialyzed 12 h against ioo volumes of the buffer, and the soluble extract used for chromatography on DEAE cellulose. The column was eluted using a gradient from o.oo5 ionic strength Tris HC1 buffer, pH 7.3 to o.o5 ionic strength Tris-HC1 buffer, pH 7.3; 200 ml of each buffer. Vertical bars indicate relative 14C activities as in Fig. I. alanine should result in the appearance of detectable radioactivity in the supernatant solution from precipitation of carboxypeptidase digested aldolase. This was tested experimentally by digestion of 35 mg of three times recrystallized aldolase from rabbit No. 3- Random substitution for tyrosine would have resulted in a total of 66 counts/min over background in the supernatant. Instead, 7 counts/min over background was found, indicating that little, if any, of the fluorophenylalanine was in place of the terminal tyrosines. The 7 counts/min could have been due to a small amount of filterable protein, or to fluorophenylalanine derived from denatured aldolase by carboxypeptidase action. Additional demonstration of phenylalanine replacement comes from a comparison of the biuret color with the 28o-m/, absorbancy for the two crystalline proteins. The biuret color is due to peptide-copper chelates and thus would be insensitive to substitution of p-fluorophenylalanine for tyrosine. Replacement of tyrosine would lower the ratio of 28o-m/, absorption to biuret color. Fig. 4 shows the relation between 28o-m/~ absorbancy and biuret color at 54 ° m/x for aldolase and
Biochim. Biophys. Acta, 54 (I96I) I45-I56
152
E. W. WESTHEAD, P. D. BOYER
glyceraldehyde dehydrogenase, together with experimental points for the fluorophenylalanine containing enzymes. The precision of the method is such that replacement to the extent of more than 3 ~0 would lead to significant deviation of the experimental points from the standard curves, whereas on the basis of radioactivity nearly 20 ~o of the tyrosine could have been replaced by the analog. '
I
'
I
'
I
'
I
Ct2
+
.
+ ~ 0.1
0.2
0.4
0.6
0.8
A 2eo rap.
Fig. 4. Relative reaction with biuret and absorbancy at 28o m# of normal and of p-fluorophenylalanine-containing aldolase (Fig. 4a) and glyceraldehyde 3-phosphate dehydrogenase (Fig. 4b). The values obtained for the A280 as measured directly are given on the abscissa, and the values obtained for the A540 in the biuret determination for the same final protein concentration are given on the ordinate. © - - © , Normal aldolase; O - - O , p-fluorophenylalanine-containing aldolase; [ ] - - •, normal glyceraldehyde 3-phosphate dehydrogenase; • - - I , p-fluorophenylalaninecontaining glyceraldehyde 3-phosphate dehydrogenase.
Effects o/p-fluorophenylalanine incorporation on protein properties Both aldolase and glyceraldehyde dehydrogenase containing p-fluorophenylalanine crystallized under the same conditions used for the crystallization of the normal enzymes. Results of measurement of enzymic activity are shown in the last line of Table I. There is no significant difference between the activities of the normal and radioactive enzymes. Chromatographic and electrophoretic separations were made as further tests of possible differences in properties of normal and p-fluorophenylalanine-containing enzymes. In addition to differences from normal enzymes, individual molecules of the aldolase and dehydrogenase preparations from experimental animals might reasonably be expected to contain different amounts of p-fluorophenylalanine. Such differences could result because following ingestion of the ration, the added p-fluorophenylalanine would likely reach the tissues before the bulk of the phenylalanine, and the rates of metabolism of phenylalanine and p-fluorophenylalanine may differ. Thus the protein synthesizing systems were likely presented with variable p-fluorophenylalanine/phenylalanine ratios through a given day. Small differences in properties reflecting differences in extent of incorporation might be detectable by the techniques used. Fig. 5 shows the result of chromatography of aldolase from rabbit No. 3 on a phosphorylated cellulose column. The protein used was the first crop of crystals from muscle extract, with specific activity within IO °/o of that shown by our purest aldolase preparations. The specific activity and specific radioactivity ratios Biochim. Biophys. +4eta, 54 (1961) 145-156
153
p-FLUOROPHENYLALANINE INCORPORATION
indicate no detectable heterogeneity with respect to fluorophenylalanine content. Chromatography of normal aldolase on phosphorylated cellulose has invariably given patterns similar to that in Fig. 5, despite the fact that chromatography on another cation exchanger, carboxymethyl cellulose yields a single peak. Rechromatography of the main peak from the elution of normal aldolase gave a single peak, indicating
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l"ig. 5. Elution p a t t e r n from c h r o m a t o g r a p h y of fluorophenylalanine-containing aldolase on p h o s p h o r y l a t e d cellulose. Crystalline aldolase from r a b b i t No. 3 was dissolved in o.o I ionic s t r e n g t h Tris HC1, p H 7.6, and adsorbed on a p h o s p h o r y l a t e d cellulose column, t h e n eluted w i t h a " l i n e a r " gradient from 75 ml of the same buffer to 75 ml of this buffer made 0. 5 ~'~ in NaC1. Vertical b a r s indicate relative 14C activities as in Fig. i.
that this heterogeneity is not an artifact of chromatography. A more complete discussion of the chromatographic behavior of normal aldolase, together with evidence for the catalytic homogeneity of the enzyme, will be given in another paper now in preparation. Fig. 6 shows the elution pattern of a sample of aldolase from rabbit, once recrystallized, from a column electrophoresis. Again, specific activity and specific radio&
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Fig. 6. E l u t i o n p a t t e r n from c o l u m n electrophoresis of fluorophenylalanine-containing aldolase. Crystalline aldolase from r a b b i t No. 3 was subjected to c o l u m n electrophoresis for 3 ° h, 35 ° V, in Tris-HC1 buffer, o.o 5 ionic strength, p H 7.8, t h e n eluted w i t h the same buffer. The broken line indicates specific enzymic activity; the triangles indicate relative 14C c o n t e n t of fractions as c o u n t s / m i n / m l of solution of u n i t a b s o r b a n c y at 280 m/~.
Biochim. Biophys. Acta, 54 (1961) 145 156
154
E. W. WESTHEAD, P, D. BOYER
activity measurements indicate no heterogeneity based on p-fluorophenylalanine content or activity. In order to determine whether or not the presence of p-fluorophenylalanine in the molecule affected the physical stability of the enzyme, a solution of four times recrystallized glyceraldehyde 3-phosphate dehydrogenase was heated until appreciable protein precipitated. The precipitate was then centrifuged down, the protein concentration of the supernatant measured, and the "C activity determined. This was repeated several times. As indicated in Fig. 7, the "C activity of the supernatant remained proportional to the protein content, showing that there was no preferential precipitation of protein with either high or low p-fluorophenylalanine content. As a further check, a sample of several times recrystallized glyceraldehyde 3-phosphate dehydrogenase from a normal rabbit was mixed with a sample of p-fluorophenylalanine containing enzyme in approximately equal amounts. Stepwise heat denaturation again failed to show any difference in stability between the normal and the p-fluorophenylalanine-protein (Fig. 7).
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Fig. 7- Stepwise heat denaturation of p-fluorophenylalanine-containing glyceraldehyde dehydrogenase. / X - - - A , Mixed sample of p-fluorophenylalanine enzyme plus normal enzyme; © - - - O , fluorophenylalanine enzyme alone. Samples were heated as described in the text, the insoluble protein removed, and the A 290 and radioactivity remaining in solution were determined. Measurements were made at A290 to avoid the absorbancy of the bound diphosphopyridine nucleotide. DISCUSSrON
The results show clearly that p-fluorophenylalanine is readily incorporated into a variety of proteins by the rabbit. The incorporation appears to occur chiefly or wholly by replacement of phenylalanine residues. The relatively high degree of incorporation found demonstrates the occurrence of little selectivity of phenylalanine over p-fluorophenylalanine by the protein synthesizing systems. A lack of prominent interference of p-fluorophenylalanine incorporation in the function of various proteins is indicated by the survival of the animal even though extensive incorporation occurs. The findings with the isolated aldolase and glyceraldehyde 3-phosphate dehydrogenase demonstrate no reduction of catalytic activity although, with aldolase, as much as 25 % of the phenylalanine residues have been replaced. Similarly, no differences in other properties of the proteins was found. Aldolase and glyceraldehyde 3-phosphate dehydrogenase fractionated and crystallized Biochim. Biophys. Acta, 54 (1961) I45-I56
p-FLUOROPHENPLALANINE INCORPORATION
155
in a normal manner, and the phenylalanine replacement did not alter the chromatographic behavior of the aldolase or the heat sensitivity of the dehydrogenase. Also, the electrophoretic pattern of blood plasma proteins was not appreciably modified. That decreased enzyme activity may accompany p-fluorophenylalanine incorporation is shown, however, b y the decrease in the activity of bacterial amylase e and penicillinase 7. The studies of RICHMOND with penicillinase 7 are particularly convincing; a high percentage of phenylalanine replacement was obtained, accompanied by a large reduction in enzyme activity and change in immunological properties. The apparently inert phenyl side chain can thus play a significant role in the tertiary structure of some proteins. The attempt to locate one or more mammalian enzymes showing critical sensitivity toward an incorporated analog may prove difficult. Perhaps a more hopeful approach, and one that is likely to be more fruitful as additional information is available about the composition of "active sites" and structural features of enzymes, is the search for specific analogs to replace amino acids known to be critical for the activity of particular enzymes. The present work was undertaken partly with this intention. A report that fluorophenylalanine replaced tyrosine in part 3°, made it appear likely that aldolase activity would be modified if fluorophenylalanine could be incorporated, since it was known that tyrosine is intimately involved with aldolase activity 23. The report was later withdrawn when the observation was found to be the result of an artifact of hydrolysis 5. The demonstration of widespread incorporation of p-fluorophenylalanine into blood, liver, and muscle proteins in about the same amounts indicates a lack of selectivity of the various protein-synthesizing systems for the natural amino acids. Some differences in extent of incorporation are to be expected because of the differences in rates of turnover of mammalian proteins. This very likely accounts for the greater degree of p-fluorophenylalanine substitution in aldolase than in glyceraldehyde 3-phosphate dehydrogenase. SIMPSON AND VELICK have shown that aldolase turns over nearly twice as fast as the dehydrogenase 29. Our data by no means proves that selectivity does not exist in some instances, and, indeed, it is possible that selectivity may be manifest when analog incorporation would result in interference in essential activity. The possibility of selectivity is indicated by the data of RABINOVITZ AND MCGRATH showing that both o-fluorophenylalanine and a-amino-fl-chlorobutyrate inhibit protein synthesis in reticulocytes but not in Ehrlich ascites cells~1. Studies with bacteria, which can be grown to many times their initial mass, are more likely to provide evidence about relative selectivity for different analogs of systems synthesizing various proteins. COWIE et al., in a series of papers, have demonstrated the relative uniformity of incorporation of a variety of analog into proteins separated on DEAE cellulose columns 4. However, despite their clear evidence that gross differences do not exist in the degrees of incorporation into a large number of protein fractions, their results do not rule out the possibility that sharp differences in degree of incorporation exist in minor components of the protein mixture. The only method of providing direct evidence about selectivity is through isolating and purifying various individual proteins as has been done by STEINBERGet al. 14, YOSHIDA AND YAMASAKI1°, and in the present work. This small amount of evidence indicates that for the 5 proteins isolated, incorporation into the individual proteins approximately parallels the incorporation into other proteins. YOSHIDAAND Y.~MASAKI Biochirn. Biophys. Acta, 54 (1961) 145-156
150
E . W . WESTHEAD, P. D. BOYER
have further been able to show that the degree of replacement of methionine by ethioDine is the same in each of a number of tryptic fragments as in the complete protein. Although our studies did not reveal any modification of enzyme activity as a result of p-fluorophenylalanine incorporation, the possibility remains open that the toxicity of p-fluorophenylalanine to mammals results largely or in part from an altered biological activity of certain proteins. Another possibility is that although an analog may perform satisfactorily in a protein, it may inhibit or produce inhibitory side products in other metabolic processes to which it is subjected. As pointed out by VAUGHAN A N D STEINBERG18, a number of analogs that have been incorporated into proteins are known to undergo other metabolic transformations and toxicity resulting from any effects on protein structure or function are often secondary to effects produced in other ways. ACKNOWLEDGEMENTS
This investigation was supported in part by U.S. Public Health Service Research Grants RG-493o and A-344I, U.S. Atomic Energy Commission Contract AT (II-I)341 and the Hill Family Foundation. REFERENCES 1 A. B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 27 (1958) 33 o. 2 G. BRAWERMAN AND M. YCAS, Arch. Biochem. Biophys., 68 (1957) 112. 3 R. S. BAKER, J. E. JOHNSON AND S. W. FOX, Biochim. Biophys. Acta, 28 (1958) 318. 4 D . B . COWIE, G. N . COHEN, E . T . BOLTON AND H . DE ROBICHON-SZULMAJSTER, Biochim. Biophys. Acta, 34 (1959) 39. 5 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 31 (1959) 378 6 A. YOSHIDA Biochim. Biophys. Acta, 41 (196o) 98. 7 M. H. RICHMOND, Biochem. J., 77 (196o) 112, 121. 8 D. B. COWIE AND G. N. COHEN, Biochim. Biophys. Acta, 26 (1957) 252. 9 D. GROS AND H. TARVER, J. Biol. Chem., 217 (1955) 169. 10 A. YOSHIDA AND M. YAMASAKI, Biochim. Biophys. Acta, 34 (1959) 158. 11 M. H. RICHMOND, BiOchem. J., 73 (1959) 261. 12 A. L. BLACK AND M. KLEIBER, J. Am. Chem. Soc., 77 (1955) 6082. 13 E. W. ~¥'ESTHEAD AND P. D. BOYER, A~$. Chem. Sac. Abstracts, September 1958 , p. 2c. 14 D . STEINBERG, M. VAUGHAN AND F. G. SHERMAN, Biochim. Biophys. Aeta, 4 ° (196o) 225. 15 j . KRUH AND J. RosA, Biochim. Biophys. Acta, 34 (1959) 561. 16 p. F. 1{RUSE, P. B. WHITE, H. A. CARTER AND T. A. McCoY, Cancer Research, 19 (1959) 122. 17 M. RABINOVITZ AND RUTH K. TUVE, Proc. Soc. Exptl. Biol. Med., IOO (1959) 222. 18 M. VAUGHAN AND D. STEINBERG, Advances in Protein Chem., Vol. 14, A c a d e m i c Press, N e w Y ork, 1959, p. 115. 19 G. N. COHEN AND F. GROS, Ann. Rev. Biochem., 29 (196o) 525 . s0 p. FLODIN AND D. V~'. KUPKE, Biochim. Biophys. Acta, 21 (1956) 368. 21 M. VAUGHAN, D. STEINBERG AND J. LOGAN, Science, 126 II957) 446. 23 j . F. TAYLOR, A. A. GREEN AND G. T. CORI, J. Biol. Chem., 173 (1948) 591 . 23 E. R. DRECHSLER, P. D. BOYER AND A. G. KOWALSKY, J. Biol. Chem., 234 (1959) 2627. 24 V. JAGANNATHAN, K. SINGH AND M. DAMODARAN, Biochem. J., 63 (1956) 9425 S. F. VELICK, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, A c a d e m i c P r e s s , N e w York, 1955, Vol. I, p. 4Ol. 26 p. D. BOYER AND A. R. SCHULZ, in R. BENESCH et al., Sulfur in Proteins, A c a d e m i c P r e s s , N e w Yo rk , 1959, p. 199. 27 E. LAYNE, in S. P. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 3, A c a d e m i c P r e s s , N e w Y o r k , 1955, p. 447. 33 M. D. ARMSTRONG AND J. D. L E w i s , J. Biol. Chem., 19o (1951) 461. 29 M. V. SIMPSON AND S. F. VELICK, J. Biol. Chem., 208 (1954) 61. 30 R. MUNIER AND G. N. COHEN, Bioehim. Biophys. Acta, 21 (1956) 592. 31 M. RABINOVlTZ AND H. MCGRATH, J. Biol. Chem., 234 (1959) 2091. 32 S. F. VELICK AND V.. RONZONI, J. Biol. Chem., 173 (1948) 627.
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