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The incorporation of β-2-thienylalanine into the β-galactosidase of Escherichia coli
108
BIOCHIMICA ET BIOPHYSICAACTA
BBA 12291 THE INCOPORATION OF fl-2-THIENYLALANINE INTO T H E fl-GALACTOSIDASE O F E S C H E R I C H I A COLI JIRI JANE~:EK* AND H. V. RICKENBERG Department of Bacteriology, Indiana University, Bloomington, Ind. (U.S.A.)
(Received April 26th, 1963)
SUMMARY The incorporation of the phenylalanine analog, fl-2-thienylalanine, into the fl-galactosidase (EC 3.2.1.23) of Escherichia coli is described. The catalytic properties of flgalactosidase in which at least 95 % of the phenylalardne residues have been replaced by the analog were apparently unchanged. Phenylalanine replacement, however, rendered the fl-galactosidase more labile than the normal enzyme to heat, urea, toluene, and trypsin (EC 3.4.4.4). Bacteria grown in the presence of fl-2-thienylalanine had lost the ability to accumulate galactosides against a concentration gradient. INTRODUCTION It has been shown 1 that a number of amino acid analogs are incorporated into protein and that in some, but not in other, cases the substitution of the natural amino acid by its analog permits the formation of functional enzymes. The incorporation of analogs of phenylalanine into proteins has been studied in microorganisms 2-4 as well as in mammals 5. For example, it was found that the phenylalanine analogs o-, m-, and p-fluorophenylalanine as well as T-ala permitted the synthesis of the enzyme alkaline phosphatase (EC 3.1.3. I) in a phenylalanine-requiring auxotroph of E. coli 3. It has also been reported that another E.coli enzyme, fl-galactosidase (EC 3.2.1.23), was synthesized in the presence of p-fluorophenylalanine, but that T-ala, although it permitted linear growth of the bacterial culture, completely inhibited induced fl-galactosidase synthesis 2. In this paper it will be shown that fl-galactosidase is in fact synthesized in the presence of T-ala under the experimental conditions employed, that T-ala is incorporated into fl-galactosidase, and that the T-ala-containing fl-galactosidase is more labile than the normal enzyme. Some of these findings have been reported in a preliminary communication 6. Abbreviations: T-ala, /?-2-thienylalamine; ONPG, o-nitrophenyl-fl-D-galactopyranoside; TMG, thiomethyl-fl-D-galactopyranoside. Permanent address : Department of Microbiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia). Biochim. Biophys. Acta, 81 (1964) Io8-I2I
~-2-THIENYLALANINE IN E. coli fl-GALACTOSIDASE
lO 9
MATERIALS AND METHODS
Bacterial strains and medium E.eoli strains ML 30, wild type, and ML 48, a phenylalanine-requiring auxotroph, were employed. Both strains form the enzyme/5-galactosidase inducibly. MONOD'Sv salts medium "56" with 0.2% glycerol as a source of C was used in all experiments. For the cultivation of strain ML 48 the medium was supplemented with nL-phenylalanine to a final concentration of 0.2 mM. Bacterial cultures were grown at 37 ° with aeration. Preparation of bacterial extracts Bacteria were harvested b y sedimentation, washed twice with 0.05 M sodium phosphate buffer (pH 7), and iesuspended in the same buffer to a density of not less than 1.2. lO TMbacteria per ml, corresponding to a protein concentration of approx. 5 nag of protein per ml. The bacteria were disrupted b y treatment for 30-40 min in a Raytheon IO kcycles magnetostrictive oscillator and the extract centrifuged for 30 min at 30 ooo x gin a refrigerated centrifuge. The precipitate was discarded and the supernate used for the assay of/5-galactosidase activity.
The induction of/5-galactosidase and its assay TMG at concentrations ranging from 0.5 mM to IO mM served as inducer of/5galactosidase./5-Galactosidase activity was determined as described earlie# with the modification that routinely/5-mercaptoethanol to a final concentration of IO mM and Mn ~+ to a final concentration of o.I mM were added to the buffer./5-Galactosidase activity is expressed as millimicromoles of ONPG hydrolyzed per min at 37 °. Specific /5-galactosidase activity is defined as the number of millimicromoles of ONPG hydrolyzed per min per mg of protein.
Other analytical methods Bacterial density was measured turbidimetrically in a Klett-Summerson colorimeter using the 66o m/~ filter and the protein content of cultures determined b y reference to a calibration curve relating bacteiial density to protein concentration. The protein content of extracts was determined b y the method of LOWRY et al. 9. For the protein determination of samples which contained/5-mercaptoethanol the LOWRY procedure was modified as follows : to 0.5 ml of the sample were added o.5 ml of 1% HgC12 and 5.0 ml of 2 % Na2CO 3 in o.175 N NaOH. From this point on the procedure was the same as that described b y LOWRY. The radioactivity of protein samples was measured in the hot trichloroacetic acid-insoluble fraction prepared b y the method of MANDELSTAM10. The trichloroacetic acid precipitate was dissolved in 0.5% NH4OH, plated on planchets, and the radioactivity determined in a gas flow counter. Self absorption was negligible at the concentrations of protein plated.
Purification of/5-galactosidase The method employed was a modification of that described b y Hu, WOLFE AND REITHEL 11. All steps were carried out at 4 °. Streptomycin sulfate at a final concentraBiochim. Biophys. Acta, 81 (1964) lO8-12i
IIO
J. JANE~EK, H. V. RICKENBERG
tion of 2.5 % was added to the crude bacterial extract containing 8-1o mg of protein per ml. The precipitate which formed was iemoved after 9 ° min b y centrifugation and discarded. The supernate was brought to 64% (NH4)2SO 4 saturation with a saturated (NH,)2SO 4 solution and kept overnight at 4 °. The precipitate was sedimented b y centrifugation and redissolved in the same buffer as that employed in the fl-galacto~idase assay. The solution was now brought to 28% (NH4)2SO 4 saturation and after 60 rain the preparation was centrifuged, the precipitate discarded and the supernate brought to 37% (NH4)zSQ saturation. After being kept overnight the precipitate which formed was removed by centrifugation, resuspended in buffer and (NH4)2SO 4 was added to a final concentration of 300/0 . After 18o min the preparation was again centrifuged and the resulting sediment dissolved in a small volume of buffer not containing any Mn 2+. At this stage of the purification procedure approximately one-half of the protein was fl-galactosidase. Starch-block electrophoresis12,13 was employed for further purification. The electrophoresis was carried out in 0.05 M sodium phosphate buffer (pH 7.6), to which fl-mercaptoethanol was added to a final concentration of io mM. The apparatus, (E.C. Apparatus Co., Swarthmore, Pa., Model EC 405) was kept at o °; the duration of individual runs varied from 20 to 30 h; 8-1o V/cm were applied. At the completion of the electrophoresis the starch was cut into sections I cm long and the proteins were eluted with 5 ml of cold fl-mercaptoethanol and Mn2+-containing buffer (pH 7) per section. The fl-galactosidase activity, protein content, and radioactivity of the supernates were then determined. After the elution of the fl-galactosidase from the starch the volume of the enzyme preparations was reduced to approx. 2 ml by dialysis against polyethylene glycol (Carbowax 4ooo), and (NH4)2SO 4 to a final saturation of 23% was added. Such preparations represented 95-1oo% pure fl-galactosidase on the basis of the comparison of their specific activities with that of a sample of crystalline E.coli, strain ML 309 fl-galactosidase. The specific activities were slightly higher than 400 ooo.
Preparation of antiserum Antiserum against crystalline fl-galactosidase was prepared b y injecting rabbits into their footpads at weekly intervals with I mg per injection of the fl-galactosidase preparation diluted in I ml of Freund's adjuvant over a 3-week period. 2 weeks after the final injections the animals were bled from their ear vein and the sera collected. The assay for the precipitation of fl-galactosidase by serum is described later and represents an adaptation of the procedure published b y COHN AND TORRIAN114.
Chemicals TMG and fl-3-thienylalanine were obtained from Mann Research Laboratories; California Corporation for Biochemical Research supplied T-ala, [i'-14Cl T-ala (specific radioactivity of 3 mC/mmole) and ONPG. [IJ4C]Lactose (specific radioactivity of I mC/mmole) was furnished by the National Bureau of Standards and TMG labeled with 14C in the thiomethyl group with a specific activity of 1.86 mC/mmole was obtained from New England Nuclear Corporation. The uniformly laC-labeled Lproline with a specific radioactivity of 5.7 mC/mmole was furnished b y Nichem Inc. Mallinckrodt potato starch was employed for the starch electrophoresis. Biochim. Biophys. Acta, 81 (1964) lO8-121
fl-2-THIENYLALANINEI1~ E, coli fl-GALACTOSIDASE
III
RESULTS
The effect of T-ala on the growth of E. colt The addition of T-ala at a concentration of I mM (final) to an exponentially growing culture of the wild-type strain resulted ill a linear rate of growth (Fig. I). Cell division was inhibited; microscopic observation and plate counts showed an insignificant change in the number of bacteria over a period of time during which the bacterial protein content of the culture doubled. Bacteria incubated in the presence of T-ala were viable as indicated b y their ability to give rise to colonies upon plating
20C
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~ 5o
Time (rain)
Fig. I. T h e effect of T - a l a on t h e g r o w t h of E. colt s t r a i n s M L 3 ° (wild type) a n d M L 48 (phenylalanine-requiring). 0 - - - 0 , M L 3o, no a d d i t i o n ; O - - O , M L 3o, x m M T-ala; I t - - m , M L 48, no a d d i t i o n ; ~ k - - A , M L 48, o.2 m M p h e n y l a l a n i n e ; E l - - D , M L 48, i m M T-ala.
on a medium not containing T-ala. The individual bacteria were larger than those of a control culture not containing the analog. The addition of T-ala to a culture of the phenylalanine auxotroph after the depletion of its intracellular phenylalanine pool b y starvation also resulted in a linear rate of growth. The growth rate of the phenylalanine auxotroph in the presence of T-ala, however, was distinctly lower than that of the wild-type strain in the presence of the same concentration of T-ala.
The induction of fl-galactosidase in the presence of T-ala i
Table I shows that the induction of fl-galactosidase b y TMG in a culture growing in the presence of I mM T-ala required higher concentrations of the inducer than did the induction of the enzyme in the absence of the analog. Table I also shows that the fl-galactosidase formed in the presence of T-ala was "cryptic"; i.e. the enzyme was poorly accessible to the substrate ONPG when fl-galactosidase activity was assayed in intact bacteria. Table I I shows that the bacteria induced in the presence of T-ala had a severely reduced capacity to concentrate lactose and TMG; i.e. a functional galactoside concentration mechanism 15 apparently was not formed during growth Biochim. Biophys. Acta, 81 (1964) lO8-121
j. JANE~EK, H. V. RICKENBERG
112
TABLE I SPECIFIC
fl-GALACTOSIDASE ACTIVITY OF E . cull ML 3 ° INDUCED WITH TMG DURING GROWTH IN T H E P R E S E N C E OF I mM T-ALA
The b a c t e r i a were g r o w n in t h e presence of T-ala a n d TMG u n t i l one d o u b l i n g of t h e b a c t e r i a l p r o t e i n h a d occurred.
T M G conch, (mM)
Specific fl-galactosidase activity of bacterial extracts
Specific fl-galactosidase activity o[ intact bacteria
0. 5 I 2. 5 5 IO
200 765 216o 38oo 48oo
4° 4° 39 4° 5°
4900
348
Growth in the absence ofT-ala."
0. 5 mM TMG
in the presence of the phenylalanine analog. A culture induced with IO mM TMG in the presence of T-ala and having a normal level of fl-galactosidase activity was unable to utilize lactose for growth, if kept in the presence of T-ala. The induction of fl-galactosidase during growth in the presence of I mM T-ala was studied in both strain ML 30 and in strain ML 48 which requires phenylalanine for TABLE II EFFECT OF
GROWTH IN
THE PRESENCE
OF
T-ALA ON GALACTOSIDE TRANSPORT
The c u l t u r e s were g r o w n in t h e presence of 2. 5 mM TMG u n t i l one d o u b l i n g of t h e b a c t e r i a l p r o t e i n h a d occurred, s e d i m e n t e d a n d t h e b a c t e r i a w a s h e d t w i c e w i t h buffer. T h e y were t h e n r e s u s p e n d e d in buffer, b r o u g h t to 37 °, aerated, a n d t h e l a c t os e or TMG a dde d. A f t e r 5 m i n i n c u b a t i o n 0. 5 ml of t h e i n c u b a t i o n m i x t u r e s were filtered t h r o u g h m e m b r a n e filters (Millipore HA, 0.45/z pore d i a m e t e r ) a n d t h e b a c t e r i a w a s h e d on t h e filter w i t h five o.2-ml p o r t i o n s of ice-cold buffer. The filter disks were t h e n g l u e d to p l a n c h e t s , a i r dried, a n d t h e r a d i o a c t i v i t y d e t e r m i n e d in a gas flow counter. The specific fl-gala c t os i da s e a c t i v i t y of t h e c u l t u r e g r o w n w i t h o u t T -ala w a s 425 o. The specific fl-galactosidase a c t i v i t y of t h e c u l t u r e g r o w n in t h e pre s e nc e of T - a l a w a s 203o. itmoles taken up per zo l~ bacteria Culture Lactose
I. I n d u c e d in presence of i mM T-ala 2. I n d u c e d in absence of T-ala
4.6 118.o
TMG
1.2 26.o
growth. TMG at a concentration of 5 mM served as inducer and o.2% glycerol as a source of C. Fig. 2 shows the differential rates of fl-galactosidase synthesis in the two strains. In the case of strain ML 48 growth ceased after the culture had gone through less than one-half doubling of the protein content (the freshly inoculated culture had a protein concentration of approx. IOO pg of protein pei ml) and fl-galactosidase was Biochim.
Biophys.
M c t a , 81 (i964) i o 8 - I 2 I
601
~-2-THIENYLALANINE IN E. coli ~-GALACTOSIDASE
113
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Growth (~g of p r o t e i n / m l ) Fig. 2. The differential rate o f #-ga]actosidase synthesis in cultures g r o w n in the presence o f
T-ala. O - - o , M L 3 o ; r q - - C ] , M L 4 8 .
synthesized at a very low differential rate. Considerable amounts of/%galactosidase, however, were formed, when the culture, after cessation of growth, was sedimented and resuspended in a medium (I mM T-ala, 5 mM TMG) from which the source of C, glycerol, had been omitted./%galactosidase synthesis under these conditions occurred in the absence of any net increase in bacterial protein. It can be seen from Fig. 3 that the synthesis of/%galactosidase in the absence of any net protein synthesis was paralleled by the incorporation of [l¢Cs]-labeled proline into the bacterial protein. Measurement of the radioactivity of the bacterial protein suggested that the differ-
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Fig. 3. The effect of an exogenous source of C on fl-galactosidase synthesis in ML 48 growing in the presence of T-ala. [3--[3, bacterial growth; A - - & , ~-galactosidase synthesis; A - - A , incorporation of [t¢C5] proline into bacterial protein.
Biochim. Bgophys. Acea, 81 (1964) lO8-121
114
j . JANE~EK, H. V. RICKENBERG
ential rate of/5-galactosidase synthesis under conditions where no net synthesis of protein occurred was about the same as the normal differential rate of synthesis in the absence of T-ala and in the presence of phenylalanine. After 17 h of incubation the specific/~-galactosidase activity of the culture corresponded to approximately one-half of the maximal specific/~-galactosidase activity of a wild-type culture grown in the absence of T-ala (or of a phenylalanine-requiring culture grown in the presence of phenylalanine).
Properties of the ~-galactosidase formed during growth in the presence of T-ala The ~-galactosidase formed in the presence of T-ala was found to be labile to a variety of reagents and conditions. Dilution: It was observed in preliminary experiments that the/~-galactosidase synthesized in the presence of T-ala was largely destroyed by the conventional toluene treatment employedin the assay of/~-galactosidase. Enzymic activity, however, could be obtained by the disruption of the bacteria byother means such as disintegration in a sonic oscillator. In order to avoid loss of activity the bacteria had to be extracted at densities corresponding to not less than 5 mg of bacterial protein per m]. Earlier experiments had shown that dilution of extracts from bacteria induced in the presence of T-ala in 0.05 M sodium phosphate buffer (pH 7) resulted in the progressive inactivation of the /~-galactosidase. This loss of enzymic activity as a result of dilution could be prevented b y the addition of - S H compounds and of Mn ~+. Maximal stability was obtained when extracts were diluted and assayed in buffer containing io mM/~-mercaptoethanol and o. I mM Mn 2+ and hence/5-galactosidase assays were carried out routinely in 0.05 M sodium phosphate buffer (pH 7) containing/5-mercaptoethanol and Mn ~+ at these concentrations. The loss of/Lgalactosidase activity resulting from the dilution of extracts in unsupplemented phosphate buffer was completely reversible. Thus an
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Fig. 4 - T h e h e a t stability of /5-galactosidase containing T-ala. Bacterial e x t r a c t s containing 250/~g of protein per ml were incubated at 5 °0 and diluted into ice-cold buffer at the times indicated. O - - O , e x t r a c t from strain ML 3 ° grown in the absence of T-ala; O - - - © , e x t r a c t from strain ML 3 ° g r o w n in the presence of I mM T-ala.
Biochim. Biophys. Acta, 81 (1964) i o 8 - I 2 I
fl-2-THIENYLALANINE IN E .
coli ~-GALACTOSIDASE
115
extract from a culture induced in the presence of T # l a was diluted to a concentration of IO ~ug of protein per ml in phosphate buffer and 90% of the fl-galactosidase activity was lost. The diluted extract was then kept for 6o rain at room temperature and at the end of this period fl-mercaptoethanol and Mn *+ were added; when the extract was now re-assayed it was found that it had recovered the specific activity of the undiluted preparation. Heat: E. coli fl-galactosidase is a relatively heat-stable enzyme 7,1~. The fl-galactosidase formed in the presence of T ala wasfound to be quite heat-labile (Fig. 4) ; most of its activity was lost after 4 min incubation at 5 °0 under conditions where normal fl-galactosidase lost no activity. The presence of a combination of fl-mercaptoethanol and Mn ~+ during the heating of the enzyme stabilized the labile fl-galactosidase completely. The heat denaturation of the fl-galactosidase formed in the presence of T-ala was irreversible. Urea: Fig. 5 shows the differential lability of normal fl-galactosidase and flgalactosidase formed in the presence of the analog to incubation with 3 M urea. fl-Mercaptoethanol and Mn *+ afforded, partial protection against inactivation; the denaturation of the enzyme by urea was irreversible. Trypsin: Normal fl-galactosidase and fl-galactosidase formed in the presence of T-ala showed significant differences with respect to their stability toward trypsin (Fig. 6), fl-Mercaptoethanol and Mn 2+ afforded only slight protection against inactivation of the enzyme by trypsin.
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1.0
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120
Time(rnin) Fig. 5. T h e s t a b i l i t y of fl-galactosidase c o n t a i n i n g T - a l a in 3 M urea. T h e i n c u b a t i o n w a s carried on a t 37 °, t h e e x t r a c t s c o n t a i n e d iooo # g of p r o t e i n per m l a n d were d i l u t e d 25-fold into buffer prior to t h e assay. 4~--@, e x t r a c t f r o m s t r a i n M L 3o g r o w n in t h e a b s e n c e of T-ala; O - - O , e x t r a c t f r o m s t r a i n M L 3o g r o w n in t h e presence of I m M T-ala.
Catalytic properties In view of the striking differences in stability between normal fl-galactosidase and fl-galactosidase synthesized in the presence of T-ala, it was of interest to see whether or not the two fl-galactosidases also differed in their catalytic properties, The pH optima of the two enzymes (crude extracts) were both at pH 6. 9 when ONPG Biochim. Biophys. Acta, 8i (1964) i o 8 - i 2 i
116
J. JANECEK, H. V. RICKENBERG 2.0
_s
:~ 1.0
P
&
J
do
~o
Time (rain)
Fig. 6. The stability of/~-galactosidase (iooo/~g of protein per ml) containing T-ala to incubation with t r y p s i n (i mg/ml) at 37 °. O - - O , e x t r a c t from strain ML 3 ° grown in the absence of T-ala; © - - O, e x t r a c t from strain ML 30 grown in the presence of I mM T-aM.
served as substrate; the activity of the/5-galactosidase formed in the presence of T-ala fell off more steeply on either side of neutrality than did the activity of normal/5galactosidase. The Km for ONPG and the Kl for lactose (determined by treating lactose as a competitive inhibitor of ONPG hydrolysis) did not differ significantly for normal/5-galactosidase and/5-galactosidase synthesized in the presence of the analog. Serology
It has been shown 1~that the incorporation of p-fluorophenylalanine into Bacillus cereus exopenicillinase changed the immunological properties of that enzyme. It has also been shown TM that the/5-galactosidase of Paracolobactrum aerogenoides which has catalytic properties indistinguishable from those of the/5-galactosidase of E.coli is 3 -~ 7 2 0 0
~o
~
5400
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3o
O_e 1800
rr
05 1.0 1.5 2.0 AntisePum added (ml)
25
Fig. 7. Precipitation of normal /~-galactosidase ( O - - O ) and /~-galactosidase containing T-ala ( O - - O) b y antiserum against crystalline/~-galactosidase. Increasing a m o u n t s of antiserum a d d e d to c o n s t a n t a m o u n t of/~-galactosidase.
Biochim. Biophys. Acta, 81 (1964) lO8-121
fl-2-THIENYLAI.ANINE IN E. coli fl-GALACTOSIDASE
117
immunologically unrelated to the E.coli fl-galactosidase. Hence it was of interest to compare the immunological behavior of normal E. coli fl-galactosidase with that of fl-galactosidase formed during growth in the presence of T-ala. Two procedures were employed in the comparison of the immunological properties of the two fl-galactosidases. In the first procedure (Fig, 7) increasing amounts of the two fl-galactosidases were added to constant amounts of antiserum; in the second procedure (Fig. 8) increasing amounts of antiserum were added to constant amounts of flgalactosidase. The antiserum, prepared ~s described under MATERIALSAND METHODS, was diluted in 0. 9 % NaC1, the fl-galactosidase preparations were in buffer containing fl-mercaptoethanol and Mn 2+. Total volumes were adjusted with buffer to 3 ml, the antiserum-fl-galactosidase mixtures incubated for 60 rain at 37 °, and then kept for 12o h at 2 °. The preparations were then centrifuged at 6000 x g for 60 min and the supernates assayed for residual fl-galactosidase activity. Enzyme preparations incubated with normal serum served as controls. (Different enzyme preparations and sera were employed in the experiments depicted in Figs. 7 and 8.) It can be seen that a given concentration of antiserum consistently precipitated somewhat more normal fl-galactosidase than fl-galactosidase formed in the presence of T-ala. The Slopes of the precipitation curves appear to be identical. One explanation for these findings
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Bo 2000
/-
o
~ ,~ looo
/°
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//° ....
I
,
1000
I
f
2000
3000
Units of ~-Galactosidase added
Fig. 8. Precipitation of normal fl-galactosidase (O---Q) and fl-galactosidase containing T-ala ( O - - O) by antiserum against crystalline fl-galactosidase. Increasing amounts of fl-galactosidase added to a constant amount of antiserum.
would be that the extract from the culture grown in the presence of the analog contained enzymically inactive but immunologically cross-reacting protein. Experiments designed to demonstrate the presence of such cross-reacting protein yielded negative results.
Evidence for the incorporation of T-ala into fl-galactosidase Evidence for the incorporation of T-aia into E. coli protein in general and flgalactosidase in particular was obtained in the following manner: Strain ML 30 was grown in a mediumcontaining I mM [I-14C]T-ala and 5 mM TMG. The culture was Biochim. Biophys. Acta, 81 (i964) lO8-121
II8
J. JANE~EK, H. V. RICKENBERG
sampled at hourly 'intervals and the radioactivity of the hot trichloroacetic acidinsoluble fraction determined. The radioactivity increased as a linear function of the increase in bacterial protein, i.e. the specific radioactivity of the newly formed protein was constant. The culture was harvested after the protein content of the culture had undergone one doubling, the bacteria disrupted, and the resulting extract submitted to electrophoresis. Fig. 9 shows that the protein fractions separated by electrophoresis
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Fig. 9. The starch electrophoresis of purified e x t r a c t of strain ML 3 ° g r o w n in the presence of I mM [IJ4C] T-ala and induced with 5 mM TMG. , protein (/,g/ml); - - , counts/ rnin per ml; . . . . . . . . . , c o u n t s / m i n per m g protein; . . . . , fl-galactosidase units/rain per ml a r b i t r a r y units.
were labeled to approximately the same extent. The specific radioactivity of the fraction showing/5-galactosidase activity was, however, considerably higher than that of the other fractions. This finding is explained by the fact that only one-half of the bacterial proteins other than /5-galactosidase were synthesized in the presence of labeled T-ala whereas most of the/5-galactosidase was formed after the addition of the labeled analog and of the inducer TMG. Chromatography and radioautography of the protein hydrolysate of the extract showed that the radioactivity of the protein was associated with T-ala only.
The extent of the replacement of phenylalanine by T-ala Essentially pure fl-galactosidase was obtained as described under MATERIALSAND METHODS.Our criterion for the purity of enzyme preparations was based on the finding that they had the same specific activity as did a sample of crystalline/5-galactosidase. The calculations determining the extent of the phenylalanine replacement were based on the published data 19 regarding the amino acid composition of the crystalline/5Biochim. Biophys. Acta, 81 (1964) i o 8 - i • i
fl-2-THIENYLALANINEIN E. col{ ~-GALACTOSIDASE
119
galactosidase from E. coli strain ML 3o9. These data show that the crystalline enzyme contains 4.73 g of phenylalanine per IOO g of crystalline/~-galactosidase. We found that in the ~-galactosidase of wild-type strain ML 30 approx. 5o % of the phenylalanine residues had been replaced by T-ala (the bacteria were harvested when one doubling of the protein content had occurred after the addition of the analog and of the inducer). In the case of the phenylalanine-requiring mutant, strain ML 48, at least 95 % of the phenylalanine residues of the/~-galactosidase had been replaced by T-ala provided the /5-galactosidase had been synthezised during growth in the presence of glycerol. If, however, glycerol was removed from the medium and ~-galactosidase synthesis occurred in the absence of any net synthesis of protein (Figs. 2 and 3), only 20% of the phenylalanine residues were replaced by T-ala. (The bacteria in this particular experiment had been incubated for I0 h in the absence of an exogenous source of C.) One assumption involved in our calculations of the replacement of phenylalanine by T-ala was that phenylalanine was the only amino acid replaced by T-ala. It has been claimed 2° that growth in the presence of I mM T-ala does not lower the tyrosine content of E. coZi protein suggesting that T-ala does not act as an analog of tyrosine. DISCUSSION
The findings presented here demonstrate clearly that/~-galactosidase is synthesized by E. coli in the presence of the phenylalanine analog, T-ala. They also show that a majority, if not all, of the phenylalanine residues of/5-galactosidase may be replaced by T-ala without interfering with the enzymic activity of this protein. Although the T-ala-containing/5-galactosidase displays apparently normal catalytic properties, it has become labile to a variety of conditions and reagents to which the/Lgalactosidase not containing any T-ala is resistant. How the replacement of phenylalanine by its analog enhances the lability of the enzyme is not clear. It has been stated 2° that growth of E. coli in the presence of T-ala does not lower the tyrosine content of the bacterial protein. However the replacement of only a few tyrosine residues by T-ala might well have escaped detection and yet sufficed to weaken the tertiary structure of protein in which replacement had occurred. In experiments done by the authors, T-ala had a slight, possibly significant, sparing effect on the growth of a tyrosinerequiring mutant when grown with limiting amounts of tyrosine~ This very slight sparing effect, even if significant, does, of course, not necessarily indicate replacement of tyrosine by T-ala in the bacterial protein. The observation that bacteria grown in the presence of T-ala have lost the ability to concentrate galactosides from the medium is analogous to the observation by COHEN AND MUNIER~ that growth in the presence of the phenylalanine analog, pfluorophenylalanine, results in the loss of galactoside "permease" activity. In this context the observation of KERRIDGE2z that Salmonella typhimurium flagella in which phenylalanine has been replaced by p-fluorophenylalanine (but not by T-ala) are nonfunctional may be of interest. The finding that strain ML 48 synthesized only minute amounts of/5-galactosidase in the presence of T-ala as long as glycerol was present in the medium is another example of the common observation that under conditions where N or a required amino acid limit growth any source of energy, utilizable by the bacteria, will suppress/~-galactosidase synthesis. After removal of glycerol, i.e., in the absence of any exogenous Biochim. Biophys. Acta, 81 (1964) lO8-i21
i2o
j. JANECEK, H. V. RICKENBERG
source of energy, fl-galactosidase was synthesized at an approximately normal differential rate. The energy required for this synthesis presumably was derived from the catabolism of pie-existing cellular material. The breakdown of pre-existing protein is further indicated by the progressively greater stability of the fl-galactosidase synthesized in the absence of an exogenous C source and by the fact that under these conditions only 20 % of the phenylalanine in fl-galactosidase is replaced by T-ala. Undoubtedly phenylalanine released as a result of the breakdown of pre-existing protein was incorporated into the fl-galactosidase formed in the absence of an exogenous source of C. The fact that COHEN AND MUNIER2 did not find any fl-galactosidase synthesis in E. coli grown in the presence of T-ala is probably exp]ained on the basis of the relatively low concentration of inducer employed by these authors and by their use of toluene in the assay plocedure. It has been shown in the experiments described in this paper that the incorporation of T-ala into E. coli protein severely diminished the ability of the bacteria to concentrate galactosides and that fl-galactosidase in which phenylalanine had been replaced by T-ala was labile to toluene. Preliminary experiments designed to examine the effect of growth in the presence of T-ala on E. coli enzymes other than fl-galactosidase indicated that hexokinase and phosphoglucomutase activity were inhibited by approx. 40% whereas Glc-6-P dehydrogenase activity was inhibited by approx. 70%. No attempt has been made to determine whether this decrease in activity is due to lowered rates of enzyme formation or whether it reflects the increased lability of the enzymes under the conditions of the assay. Experiments in which bacteria were grown and induced in the presence of fi-3thienylalanine 2. yielded results qualitatively similar to those reported for T-ala; it appeared that the effect of fl-3-thienylalanine was less severe, both with respect to the inhibition of growth and the lability of the fl-galactosidase formed than was the effect of T-ala. However, no attempt was made to examine the effect of/5-3-thienylalanine incorporation in more detail. ACKNOWLEDGEMENTS
We should like to thank Professor K. Wallenfels of Freiburg University for a generous supply of crystalline E. coli, Strain ML 309 fl-galactosidase. We should like to thank Mrs. W. STERN and Mrs. P. ASHMAN for their excellent technical assistance. The investigations described here were supported by research grants from the National Science Foundation (G 15037) and the Public Health Service (A-5868). REFERENCES 1 G. N. COHEN AND F. GROS, Ann. Rev. Biochem., 29 (196o) 525 • i~ G. N. COHEN AND R. MUNIER, Biochim. Biophys. Acta, 31 (1959) 347. 8 R. L. MUNIER, Compt. Rend., 250 (196o) 3524. 4 M. H. RICHMOND, Biochem. J., 77 (196o) 121. s E. W. WESTHEAD AND P. D. BOYER, Biochim. Biophys. Acta, 54 (1961) 144. J. JANECEK AND H. V. RICKENBERG, Bacteriol. Proc., (1962) lO5. M. COHN AND J. MONOD, Biochim. Biophys. Acta, 7 (1951) 153. 8 H. V. RICKENBERG, C. YANOFSKY AND D. M. BONNER, J. Bacteriol., 66 (1953) 683. O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, ,[. Biol. Chem., 193 (I95 I) 265.
Biochim. Biophys. Acta, 8i (1964) lO8-12i
fl-2-THIENYLALANINEIN E.
coli fl-GALACTOSIDASE
12I
10 j . MANDELSTAM, Biochem. J., 69 (1958) IiO. n A. S. L. H u , R. G. WOLFE AND F. J. REITHEL, Arch. Biochem. Biophys., 81 (1959) 500. 12 H. G. KUNKEL AND R. J. SLATER, Proc. Soc. Exptl. Biol. Med., 80 (1952) 42. 13 B. ROTMAN AND S. SPIEGELMANN, J. Bacteriol, 68 (1954) 419 • 14 M. COHN AND A. M. TORRIANI, J. Immunol., 69 (1952) 471. is H. V. RICKENBERG, G. IxT. COHEN, G. BUTTIN AND J. IV[ONOD, Ann. Inst. Pasteur, 91 (1956) 829. is M. L. ZARNITZ, Dissertation, F r e i b u r g U n i v e r s i t y (Germa ny), 1958. 17 M. H. RICHMOND, Biochem, J., 77 (196o) 112. 18 j . ~q~. ANDERSON AND H. V. RICKENBERG, J. Bacteriol., 80 (196o) 297. 19 K. WALLENFELS AND A. ARENS, Biochem. Z., 332 (196o) 247. 20 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 31 (1959) 378. 2x D. KERRIDGE, Biochim. Biophys. Acta, 31 (1959) 579. ~2 R. L. MUNIER AND G. SARRAZIN, Compt. Rend., 254 (1962) 2853.
Biochim. Biophys. Acta, 81 (i964) i o 8 - i 2 i
BIOCHIMICA ET BIOPHYSICAACTA
BBA 12291 THE INCOPORATION OF fl-2-THIENYLALANINE INTO T H E fl-GALACTOSIDASE O F E S C H E R I C H I A COLI JIRI JANE~:EK* AND H. V. RICKENBERG Department of Bacteriology, Indiana University, Bloomington, Ind. (U.S.A.)
(Received April 26th, 1963)
SUMMARY The incorporation of the phenylalanine analog, fl-2-thienylalanine, into the fl-galactosidase (EC 3.2.1.23) of Escherichia coli is described. The catalytic properties of flgalactosidase in which at least 95 % of the phenylalardne residues have been replaced by the analog were apparently unchanged. Phenylalanine replacement, however, rendered the fl-galactosidase more labile than the normal enzyme to heat, urea, toluene, and trypsin (EC 3.4.4.4). Bacteria grown in the presence of fl-2-thienylalanine had lost the ability to accumulate galactosides against a concentration gradient. INTRODUCTION It has been shown 1 that a number of amino acid analogs are incorporated into protein and that in some, but not in other, cases the substitution of the natural amino acid by its analog permits the formation of functional enzymes. The incorporation of analogs of phenylalanine into proteins has been studied in microorganisms 2-4 as well as in mammals 5. For example, it was found that the phenylalanine analogs o-, m-, and p-fluorophenylalanine as well as T-ala permitted the synthesis of the enzyme alkaline phosphatase (EC 3.1.3. I) in a phenylalanine-requiring auxotroph of E. coli 3. It has also been reported that another E.coli enzyme, fl-galactosidase (EC 3.2.1.23), was synthesized in the presence of p-fluorophenylalanine, but that T-ala, although it permitted linear growth of the bacterial culture, completely inhibited induced fl-galactosidase synthesis 2. In this paper it will be shown that fl-galactosidase is in fact synthesized in the presence of T-ala under the experimental conditions employed, that T-ala is incorporated into fl-galactosidase, and that the T-ala-containing fl-galactosidase is more labile than the normal enzyme. Some of these findings have been reported in a preliminary communication 6. Abbreviations: T-ala, /?-2-thienylalamine; ONPG, o-nitrophenyl-fl-D-galactopyranoside; TMG, thiomethyl-fl-D-galactopyranoside. Permanent address : Department of Microbiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia). Biochim. Biophys. Acta, 81 (1964) Io8-I2I
~-2-THIENYLALANINE IN E. coli fl-GALACTOSIDASE
lO 9
MATERIALS AND METHODS
Bacterial strains and medium E.eoli strains ML 30, wild type, and ML 48, a phenylalanine-requiring auxotroph, were employed. Both strains form the enzyme/5-galactosidase inducibly. MONOD'Sv salts medium "56" with 0.2% glycerol as a source of C was used in all experiments. For the cultivation of strain ML 48 the medium was supplemented with nL-phenylalanine to a final concentration of 0.2 mM. Bacterial cultures were grown at 37 ° with aeration. Preparation of bacterial extracts Bacteria were harvested b y sedimentation, washed twice with 0.05 M sodium phosphate buffer (pH 7), and iesuspended in the same buffer to a density of not less than 1.2. lO TMbacteria per ml, corresponding to a protein concentration of approx. 5 nag of protein per ml. The bacteria were disrupted b y treatment for 30-40 min in a Raytheon IO kcycles magnetostrictive oscillator and the extract centrifuged for 30 min at 30 ooo x gin a refrigerated centrifuge. The precipitate was discarded and the supernate used for the assay of/5-galactosidase activity.
The induction of/5-galactosidase and its assay TMG at concentrations ranging from 0.5 mM to IO mM served as inducer of/5galactosidase./5-Galactosidase activity was determined as described earlie# with the modification that routinely/5-mercaptoethanol to a final concentration of IO mM and Mn ~+ to a final concentration of o.I mM were added to the buffer./5-Galactosidase activity is expressed as millimicromoles of ONPG hydrolyzed per min at 37 °. Specific /5-galactosidase activity is defined as the number of millimicromoles of ONPG hydrolyzed per min per mg of protein.
Other analytical methods Bacterial density was measured turbidimetrically in a Klett-Summerson colorimeter using the 66o m/~ filter and the protein content of cultures determined b y reference to a calibration curve relating bacteiial density to protein concentration. The protein content of extracts was determined b y the method of LOWRY et al. 9. For the protein determination of samples which contained/5-mercaptoethanol the LOWRY procedure was modified as follows : to 0.5 ml of the sample were added o.5 ml of 1% HgC12 and 5.0 ml of 2 % Na2CO 3 in o.175 N NaOH. From this point on the procedure was the same as that described b y LOWRY. The radioactivity of protein samples was measured in the hot trichloroacetic acid-insoluble fraction prepared b y the method of MANDELSTAM10. The trichloroacetic acid precipitate was dissolved in 0.5% NH4OH, plated on planchets, and the radioactivity determined in a gas flow counter. Self absorption was negligible at the concentrations of protein plated.
Purification of/5-galactosidase The method employed was a modification of that described b y Hu, WOLFE AND REITHEL 11. All steps were carried out at 4 °. Streptomycin sulfate at a final concentraBiochim. Biophys. Acta, 81 (1964) lO8-12i
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J. JANE~EK, H. V. RICKENBERG
tion of 2.5 % was added to the crude bacterial extract containing 8-1o mg of protein per ml. The precipitate which formed was iemoved after 9 ° min b y centrifugation and discarded. The supernate was brought to 64% (NH4)2SO 4 saturation with a saturated (NH,)2SO 4 solution and kept overnight at 4 °. The precipitate was sedimented b y centrifugation and redissolved in the same buffer as that employed in the fl-galacto~idase assay. The solution was now brought to 28% (NH4)2SO 4 saturation and after 60 rain the preparation was centrifuged, the precipitate discarded and the supernate brought to 37% (NH4)zSQ saturation. After being kept overnight the precipitate which formed was removed by centrifugation, resuspended in buffer and (NH4)2SO 4 was added to a final concentration of 300/0 . After 18o min the preparation was again centrifuged and the resulting sediment dissolved in a small volume of buffer not containing any Mn 2+. At this stage of the purification procedure approximately one-half of the protein was fl-galactosidase. Starch-block electrophoresis12,13 was employed for further purification. The electrophoresis was carried out in 0.05 M sodium phosphate buffer (pH 7.6), to which fl-mercaptoethanol was added to a final concentration of io mM. The apparatus, (E.C. Apparatus Co., Swarthmore, Pa., Model EC 405) was kept at o °; the duration of individual runs varied from 20 to 30 h; 8-1o V/cm were applied. At the completion of the electrophoresis the starch was cut into sections I cm long and the proteins were eluted with 5 ml of cold fl-mercaptoethanol and Mn2+-containing buffer (pH 7) per section. The fl-galactosidase activity, protein content, and radioactivity of the supernates were then determined. After the elution of the fl-galactosidase from the starch the volume of the enzyme preparations was reduced to approx. 2 ml by dialysis against polyethylene glycol (Carbowax 4ooo), and (NH4)2SO 4 to a final saturation of 23% was added. Such preparations represented 95-1oo% pure fl-galactosidase on the basis of the comparison of their specific activities with that of a sample of crystalline E.coli, strain ML 309 fl-galactosidase. The specific activities were slightly higher than 400 ooo.
Preparation of antiserum Antiserum against crystalline fl-galactosidase was prepared b y injecting rabbits into their footpads at weekly intervals with I mg per injection of the fl-galactosidase preparation diluted in I ml of Freund's adjuvant over a 3-week period. 2 weeks after the final injections the animals were bled from their ear vein and the sera collected. The assay for the precipitation of fl-galactosidase by serum is described later and represents an adaptation of the procedure published b y COHN AND TORRIAN114.
Chemicals TMG and fl-3-thienylalanine were obtained from Mann Research Laboratories; California Corporation for Biochemical Research supplied T-ala, [i'-14Cl T-ala (specific radioactivity of 3 mC/mmole) and ONPG. [IJ4C]Lactose (specific radioactivity of I mC/mmole) was furnished by the National Bureau of Standards and TMG labeled with 14C in the thiomethyl group with a specific activity of 1.86 mC/mmole was obtained from New England Nuclear Corporation. The uniformly laC-labeled Lproline with a specific radioactivity of 5.7 mC/mmole was furnished b y Nichem Inc. Mallinckrodt potato starch was employed for the starch electrophoresis. Biochim. Biophys. Acta, 81 (1964) lO8-121
fl-2-THIENYLALANINEI1~ E, coli fl-GALACTOSIDASE
III
RESULTS
The effect of T-ala on the growth of E. colt The addition of T-ala at a concentration of I mM (final) to an exponentially growing culture of the wild-type strain resulted ill a linear rate of growth (Fig. I). Cell division was inhibited; microscopic observation and plate counts showed an insignificant change in the number of bacteria over a period of time during which the bacterial protein content of the culture doubled. Bacteria incubated in the presence of T-ala were viable as indicated b y their ability to give rise to colonies upon plating
20C
r~
l:zI 0 0
I_
~ 5o
Time (rain)
Fig. I. T h e effect of T - a l a on t h e g r o w t h of E. colt s t r a i n s M L 3 ° (wild type) a n d M L 48 (phenylalanine-requiring). 0 - - - 0 , M L 3o, no a d d i t i o n ; O - - O , M L 3o, x m M T-ala; I t - - m , M L 48, no a d d i t i o n ; ~ k - - A , M L 48, o.2 m M p h e n y l a l a n i n e ; E l - - D , M L 48, i m M T-ala.
on a medium not containing T-ala. The individual bacteria were larger than those of a control culture not containing the analog. The addition of T-ala to a culture of the phenylalanine auxotroph after the depletion of its intracellular phenylalanine pool b y starvation also resulted in a linear rate of growth. The growth rate of the phenylalanine auxotroph in the presence of T-ala, however, was distinctly lower than that of the wild-type strain in the presence of the same concentration of T-ala.
The induction of fl-galactosidase in the presence of T-ala i
Table I shows that the induction of fl-galactosidase b y TMG in a culture growing in the presence of I mM T-ala required higher concentrations of the inducer than did the induction of the enzyme in the absence of the analog. Table I also shows that the fl-galactosidase formed in the presence of T-ala was "cryptic"; i.e. the enzyme was poorly accessible to the substrate ONPG when fl-galactosidase activity was assayed in intact bacteria. Table I I shows that the bacteria induced in the presence of T-ala had a severely reduced capacity to concentrate lactose and TMG; i.e. a functional galactoside concentration mechanism 15 apparently was not formed during growth Biochim. Biophys. Acta, 81 (1964) lO8-121
j. JANE~EK, H. V. RICKENBERG
112
TABLE I SPECIFIC
fl-GALACTOSIDASE ACTIVITY OF E . cull ML 3 ° INDUCED WITH TMG DURING GROWTH IN T H E P R E S E N C E OF I mM T-ALA
The b a c t e r i a were g r o w n in t h e presence of T-ala a n d TMG u n t i l one d o u b l i n g of t h e b a c t e r i a l p r o t e i n h a d occurred.
T M G conch, (mM)
Specific fl-galactosidase activity of bacterial extracts
Specific fl-galactosidase activity o[ intact bacteria
0. 5 I 2. 5 5 IO
200 765 216o 38oo 48oo
4° 4° 39 4° 5°
4900
348
Growth in the absence ofT-ala."
0. 5 mM TMG
in the presence of the phenylalanine analog. A culture induced with IO mM TMG in the presence of T-ala and having a normal level of fl-galactosidase activity was unable to utilize lactose for growth, if kept in the presence of T-ala. The induction of fl-galactosidase during growth in the presence of I mM T-ala was studied in both strain ML 30 and in strain ML 48 which requires phenylalanine for TABLE II EFFECT OF
GROWTH IN
THE PRESENCE
OF
T-ALA ON GALACTOSIDE TRANSPORT
The c u l t u r e s were g r o w n in t h e presence of 2. 5 mM TMG u n t i l one d o u b l i n g of t h e b a c t e r i a l p r o t e i n h a d occurred, s e d i m e n t e d a n d t h e b a c t e r i a w a s h e d t w i c e w i t h buffer. T h e y were t h e n r e s u s p e n d e d in buffer, b r o u g h t to 37 °, aerated, a n d t h e l a c t os e or TMG a dde d. A f t e r 5 m i n i n c u b a t i o n 0. 5 ml of t h e i n c u b a t i o n m i x t u r e s were filtered t h r o u g h m e m b r a n e filters (Millipore HA, 0.45/z pore d i a m e t e r ) a n d t h e b a c t e r i a w a s h e d on t h e filter w i t h five o.2-ml p o r t i o n s of ice-cold buffer. The filter disks were t h e n g l u e d to p l a n c h e t s , a i r dried, a n d t h e r a d i o a c t i v i t y d e t e r m i n e d in a gas flow counter. The specific fl-gala c t os i da s e a c t i v i t y of t h e c u l t u r e g r o w n w i t h o u t T -ala w a s 425 o. The specific fl-galactosidase a c t i v i t y of t h e c u l t u r e g r o w n in t h e pre s e nc e of T - a l a w a s 203o. itmoles taken up per zo l~ bacteria Culture Lactose
I. I n d u c e d in presence of i mM T-ala 2. I n d u c e d in absence of T-ala
4.6 118.o
TMG
1.2 26.o
growth. TMG at a concentration of 5 mM served as inducer and o.2% glycerol as a source of C. Fig. 2 shows the differential rates of fl-galactosidase synthesis in the two strains. In the case of strain ML 48 growth ceased after the culture had gone through less than one-half doubling of the protein content (the freshly inoculated culture had a protein concentration of approx. IOO pg of protein pei ml) and fl-galactosidase was Biochim.
Biophys.
M c t a , 81 (i964) i o 8 - I 2 I
601
~-2-THIENYLALANINE IN E. coli ~-GALACTOSIDASE
113
/o
i
/
~az
oOo4
l /
1°°I / I c6
2o
4~---; 6o
~o
60
Growth (~g of p r o t e i n / m l ) Fig. 2. The differential rate o f #-ga]actosidase synthesis in cultures g r o w n in the presence o f
T-ala. O - - o , M L 3 o ; r q - - C ] , M L 4 8 .
synthesized at a very low differential rate. Considerable amounts of/%galactosidase, however, were formed, when the culture, after cessation of growth, was sedimented and resuspended in a medium (I mM T-ala, 5 mM TMG) from which the source of C, glycerol, had been omitted./%galactosidase synthesis under these conditions occurred in the absence of any net increase in bacterial protein. It can be seen from Fig. 3 that the synthesis of/%galactosidase in the absence of any net protein synthesis was paralleled by the incorporation of [l¢Cs]-labeled proline into the bacterial protein. Measurement of the radioactivity of the bacterial protein suggested that the differ-
3
i "6- ~ 8 ~5o ®~~ -80 '-o E
\
~
C
~ "6
-o ~
~2oc
tt
"6
~
•$,
-6 _n.--.--'-~
"~"0
60
~'
~
.o, ~, 6 0 o = / : t l o o ~G = .-= ~
/ I -~ J 07 / / - 71 ,Eo z
120 180 60 Time(rnin)
/
~ -~
120
~.
4o
,~_
2o-~ 8 E .o
S .E
Fig. 3. The effect of an exogenous source of C on fl-galactosidase synthesis in ML 48 growing in the presence of T-ala. [3--[3, bacterial growth; A - - & , ~-galactosidase synthesis; A - - A , incorporation of [t¢C5] proline into bacterial protein.
Biochim. Bgophys. Acea, 81 (1964) lO8-121
114
j . JANE~EK, H. V. RICKENBERG
ential rate of/5-galactosidase synthesis under conditions where no net synthesis of protein occurred was about the same as the normal differential rate of synthesis in the absence of T-ala and in the presence of phenylalanine. After 17 h of incubation the specific/~-galactosidase activity of the culture corresponded to approximately one-half of the maximal specific/~-galactosidase activity of a wild-type culture grown in the absence of T-ala (or of a phenylalanine-requiring culture grown in the presence of phenylalanine).
Properties of the ~-galactosidase formed during growth in the presence of T-ala The ~-galactosidase formed in the presence of T-ala was found to be labile to a variety of reagents and conditions. Dilution: It was observed in preliminary experiments that the/~-galactosidase synthesized in the presence of T-ala was largely destroyed by the conventional toluene treatment employedin the assay of/~-galactosidase. Enzymic activity, however, could be obtained by the disruption of the bacteria byother means such as disintegration in a sonic oscillator. In order to avoid loss of activity the bacteria had to be extracted at densities corresponding to not less than 5 mg of bacterial protein per m]. Earlier experiments had shown that dilution of extracts from bacteria induced in the presence of T-ala in 0.05 M sodium phosphate buffer (pH 7) resulted in the progressive inactivation of the /~-galactosidase. This loss of enzymic activity as a result of dilution could be prevented b y the addition of - S H compounds and of Mn ~+. Maximal stability was obtained when extracts were diluted and assayed in buffer containing io mM/~-mercaptoethanol and o. I mM Mn 2+ and hence/5-galactosidase assays were carried out routinely in 0.05 M sodium phosphate buffer (pH 7) containing/5-mercaptoethanol and Mn ~+ at these concentrations. The loss of/Lgalactosidase activity resulting from the dilution of extracts in unsupplemented phosphate buffer was completely reversible. Thus an
2.0
•
•
_
•
•
~1.C a)
o~
_J
~o 3
6
9
12
Time (rain)
Fig. 4 - T h e h e a t stability of /5-galactosidase containing T-ala. Bacterial e x t r a c t s containing 250/~g of protein per ml were incubated at 5 °0 and diluted into ice-cold buffer at the times indicated. O - - O , e x t r a c t from strain ML 3 ° grown in the absence of T-ala; O - - - © , e x t r a c t from strain ML 3 ° g r o w n in the presence of I mM T-ala.
Biochim. Biophys. Acta, 81 (1964) i o 8 - I 2 I
fl-2-THIENYLALANINE IN E .
coli ~-GALACTOSIDASE
115
extract from a culture induced in the presence of T # l a was diluted to a concentration of IO ~ug of protein per ml in phosphate buffer and 90% of the fl-galactosidase activity was lost. The diluted extract was then kept for 6o rain at room temperature and at the end of this period fl-mercaptoethanol and Mn *+ were added; when the extract was now re-assayed it was found that it had recovered the specific activity of the undiluted preparation. Heat: E. coli fl-galactosidase is a relatively heat-stable enzyme 7,1~. The fl-galactosidase formed in the presence of T ala wasfound to be quite heat-labile (Fig. 4) ; most of its activity was lost after 4 min incubation at 5 °0 under conditions where normal fl-galactosidase lost no activity. The presence of a combination of fl-mercaptoethanol and Mn ~+ during the heating of the enzyme stabilized the labile fl-galactosidase completely. The heat denaturation of the fl-galactosidase formed in the presence of T-ala was irreversible. Urea: Fig. 5 shows the differential lability of normal fl-galactosidase and flgalactosidase formed in the presence of the analog to incubation with 3 M urea. fl-Mercaptoethanol and Mn *+ afforded, partial protection against inactivation; the denaturation of the enzyme by urea was irreversible. Trypsin: Normal fl-galactosidase and fl-galactosidase formed in the presence of T-ala showed significant differences with respect to their stability toward trypsin (Fig. 6), fl-Mercaptoethanol and Mn 2+ afforded only slight protection against inactivation of the enzyme by trypsin.
2.0
•
@
•
•
._>
1.0
~ o
"-O
~o
120
Time(rnin) Fig. 5. T h e s t a b i l i t y of fl-galactosidase c o n t a i n i n g T - a l a in 3 M urea. T h e i n c u b a t i o n w a s carried on a t 37 °, t h e e x t r a c t s c o n t a i n e d iooo # g of p r o t e i n per m l a n d were d i l u t e d 25-fold into buffer prior to t h e assay. 4~--@, e x t r a c t f r o m s t r a i n M L 3o g r o w n in t h e a b s e n c e of T-ala; O - - O , e x t r a c t f r o m s t r a i n M L 3o g r o w n in t h e presence of I m M T-ala.
Catalytic properties In view of the striking differences in stability between normal fl-galactosidase and fl-galactosidase synthesized in the presence of T-ala, it was of interest to see whether or not the two fl-galactosidases also differed in their catalytic properties, The pH optima of the two enzymes (crude extracts) were both at pH 6. 9 when ONPG Biochim. Biophys. Acta, 8i (1964) i o 8 - i 2 i
116
J. JANECEK, H. V. RICKENBERG 2.0
_s
:~ 1.0
P
&
J
do
~o
Time (rain)
Fig. 6. The stability of/~-galactosidase (iooo/~g of protein per ml) containing T-ala to incubation with t r y p s i n (i mg/ml) at 37 °. O - - O , e x t r a c t from strain ML 3 ° grown in the absence of T-ala; © - - O, e x t r a c t from strain ML 30 grown in the presence of I mM T-aM.
served as substrate; the activity of the/5-galactosidase formed in the presence of T-ala fell off more steeply on either side of neutrality than did the activity of normal/5galactosidase. The Km for ONPG and the Kl for lactose (determined by treating lactose as a competitive inhibitor of ONPG hydrolysis) did not differ significantly for normal/5-galactosidase and/5-galactosidase synthesized in the presence of the analog. Serology
It has been shown 1~that the incorporation of p-fluorophenylalanine into Bacillus cereus exopenicillinase changed the immunological properties of that enzyme. It has also been shown TM that the/5-galactosidase of Paracolobactrum aerogenoides which has catalytic properties indistinguishable from those of the/5-galactosidase of E.coli is 3 -~ 7 2 0 0
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~
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3o
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rr
05 1.0 1.5 2.0 AntisePum added (ml)
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Fig. 7. Precipitation of normal /~-galactosidase ( O - - O ) and /~-galactosidase containing T-ala ( O - - O) b y antiserum against crystalline/~-galactosidase. Increasing a m o u n t s of antiserum a d d e d to c o n s t a n t a m o u n t of/~-galactosidase.
Biochim. Biophys. Acta, 81 (1964) lO8-121
fl-2-THIENYLAI.ANINE IN E. coli fl-GALACTOSIDASE
117
immunologically unrelated to the E.coli fl-galactosidase. Hence it was of interest to compare the immunological behavior of normal E. coli fl-galactosidase with that of fl-galactosidase formed during growth in the presence of T-ala. Two procedures were employed in the comparison of the immunological properties of the two fl-galactosidases. In the first procedure (Fig, 7) increasing amounts of the two fl-galactosidases were added to constant amounts of antiserum; in the second procedure (Fig. 8) increasing amounts of antiserum were added to constant amounts of flgalactosidase. The antiserum, prepared ~s described under MATERIALSAND METHODS, was diluted in 0. 9 % NaC1, the fl-galactosidase preparations were in buffer containing fl-mercaptoethanol and Mn 2+. Total volumes were adjusted with buffer to 3 ml, the antiserum-fl-galactosidase mixtures incubated for 60 rain at 37 °, and then kept for 12o h at 2 °. The preparations were then centrifuged at 6000 x g for 60 min and the supernates assayed for residual fl-galactosidase activity. Enzyme preparations incubated with normal serum served as controls. (Different enzyme preparations and sera were employed in the experiments depicted in Figs. 7 and 8.) It can be seen that a given concentration of antiserum consistently precipitated somewhat more normal fl-galactosidase than fl-galactosidase formed in the presence of T-ala. The Slopes of the precipitation curves appear to be identical. One explanation for these findings
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,
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Fig. 8. Precipitation of normal fl-galactosidase (O---Q) and fl-galactosidase containing T-ala ( O - - O) by antiserum against crystalline fl-galactosidase. Increasing amounts of fl-galactosidase added to a constant amount of antiserum.
would be that the extract from the culture grown in the presence of the analog contained enzymically inactive but immunologically cross-reacting protein. Experiments designed to demonstrate the presence of such cross-reacting protein yielded negative results.
Evidence for the incorporation of T-ala into fl-galactosidase Evidence for the incorporation of T-aia into E. coli protein in general and flgalactosidase in particular was obtained in the following manner: Strain ML 30 was grown in a mediumcontaining I mM [I-14C]T-ala and 5 mM TMG. The culture was Biochim. Biophys. Acta, 81 (i964) lO8-121
II8
J. JANE~EK, H. V. RICKENBERG
sampled at hourly 'intervals and the radioactivity of the hot trichloroacetic acidinsoluble fraction determined. The radioactivity increased as a linear function of the increase in bacterial protein, i.e. the specific radioactivity of the newly formed protein was constant. The culture was harvested after the protein content of the culture had undergone one doubling, the bacteria disrupted, and the resulting extract submitted to electrophoresis. Fig. 9 shows that the protein fractions separated by electrophoresis
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Fig. 9. The starch electrophoresis of purified e x t r a c t of strain ML 3 ° g r o w n in the presence of I mM [IJ4C] T-ala and induced with 5 mM TMG. , protein (/,g/ml); - - , counts/ rnin per ml; . . . . . . . . . , c o u n t s / m i n per m g protein; . . . . , fl-galactosidase units/rain per ml a r b i t r a r y units.
were labeled to approximately the same extent. The specific radioactivity of the fraction showing/5-galactosidase activity was, however, considerably higher than that of the other fractions. This finding is explained by the fact that only one-half of the bacterial proteins other than /5-galactosidase were synthesized in the presence of labeled T-ala whereas most of the/5-galactosidase was formed after the addition of the labeled analog and of the inducer TMG. Chromatography and radioautography of the protein hydrolysate of the extract showed that the radioactivity of the protein was associated with T-ala only.
The extent of the replacement of phenylalanine by T-ala Essentially pure fl-galactosidase was obtained as described under MATERIALSAND METHODS.Our criterion for the purity of enzyme preparations was based on the finding that they had the same specific activity as did a sample of crystalline/5-galactosidase. The calculations determining the extent of the phenylalanine replacement were based on the published data 19 regarding the amino acid composition of the crystalline/5Biochim. Biophys. Acta, 81 (1964) i o 8 - i • i
fl-2-THIENYLALANINEIN E. col{ ~-GALACTOSIDASE
119
galactosidase from E. coli strain ML 3o9. These data show that the crystalline enzyme contains 4.73 g of phenylalanine per IOO g of crystalline/~-galactosidase. We found that in the ~-galactosidase of wild-type strain ML 30 approx. 5o % of the phenylalanine residues had been replaced by T-ala (the bacteria were harvested when one doubling of the protein content had occurred after the addition of the analog and of the inducer). In the case of the phenylalanine-requiring mutant, strain ML 48, at least 95 % of the phenylalanine residues of the/~-galactosidase had been replaced by T-ala provided the /5-galactosidase had been synthezised during growth in the presence of glycerol. If, however, glycerol was removed from the medium and ~-galactosidase synthesis occurred in the absence of any net synthesis of protein (Figs. 2 and 3), only 20% of the phenylalanine residues were replaced by T-ala. (The bacteria in this particular experiment had been incubated for I0 h in the absence of an exogenous source of C.) One assumption involved in our calculations of the replacement of phenylalanine by T-ala was that phenylalanine was the only amino acid replaced by T-ala. It has been claimed 2° that growth in the presence of I mM T-ala does not lower the tyrosine content of E. coZi protein suggesting that T-ala does not act as an analog of tyrosine. DISCUSSION
The findings presented here demonstrate clearly that/~-galactosidase is synthesized by E. coli in the presence of the phenylalanine analog, T-ala. They also show that a majority, if not all, of the phenylalanine residues of/5-galactosidase may be replaced by T-ala without interfering with the enzymic activity of this protein. Although the T-ala-containing/5-galactosidase displays apparently normal catalytic properties, it has become labile to a variety of conditions and reagents to which the/Lgalactosidase not containing any T-ala is resistant. How the replacement of phenylalanine by its analog enhances the lability of the enzyme is not clear. It has been stated 2° that growth of E. coli in the presence of T-ala does not lower the tyrosine content of the bacterial protein. However the replacement of only a few tyrosine residues by T-ala might well have escaped detection and yet sufficed to weaken the tertiary structure of protein in which replacement had occurred. In experiments done by the authors, T-ala had a slight, possibly significant, sparing effect on the growth of a tyrosinerequiring mutant when grown with limiting amounts of tyrosine~ This very slight sparing effect, even if significant, does, of course, not necessarily indicate replacement of tyrosine by T-ala in the bacterial protein. The observation that bacteria grown in the presence of T-ala have lost the ability to concentrate galactosides from the medium is analogous to the observation by COHEN AND MUNIER~ that growth in the presence of the phenylalanine analog, pfluorophenylalanine, results in the loss of galactoside "permease" activity. In this context the observation of KERRIDGE2z that Salmonella typhimurium flagella in which phenylalanine has been replaced by p-fluorophenylalanine (but not by T-ala) are nonfunctional may be of interest. The finding that strain ML 48 synthesized only minute amounts of/5-galactosidase in the presence of T-ala as long as glycerol was present in the medium is another example of the common observation that under conditions where N or a required amino acid limit growth any source of energy, utilizable by the bacteria, will suppress/~-galactosidase synthesis. After removal of glycerol, i.e., in the absence of any exogenous Biochim. Biophys. Acta, 81 (1964) lO8-i21
i2o
j. JANECEK, H. V. RICKENBERG
source of energy, fl-galactosidase was synthesized at an approximately normal differential rate. The energy required for this synthesis presumably was derived from the catabolism of pie-existing cellular material. The breakdown of pre-existing protein is further indicated by the progressively greater stability of the fl-galactosidase synthesized in the absence of an exogenous C source and by the fact that under these conditions only 20 % of the phenylalanine in fl-galactosidase is replaced by T-ala. Undoubtedly phenylalanine released as a result of the breakdown of pre-existing protein was incorporated into the fl-galactosidase formed in the absence of an exogenous source of C. The fact that COHEN AND MUNIER2 did not find any fl-galactosidase synthesis in E. coli grown in the presence of T-ala is probably exp]ained on the basis of the relatively low concentration of inducer employed by these authors and by their use of toluene in the assay plocedure. It has been shown in the experiments described in this paper that the incorporation of T-ala into E. coli protein severely diminished the ability of the bacteria to concentrate galactosides and that fl-galactosidase in which phenylalanine had been replaced by T-ala was labile to toluene. Preliminary experiments designed to examine the effect of growth in the presence of T-ala on E. coli enzymes other than fl-galactosidase indicated that hexokinase and phosphoglucomutase activity were inhibited by approx. 40% whereas Glc-6-P dehydrogenase activity was inhibited by approx. 70%. No attempt has been made to determine whether this decrease in activity is due to lowered rates of enzyme formation or whether it reflects the increased lability of the enzymes under the conditions of the assay. Experiments in which bacteria were grown and induced in the presence of fi-3thienylalanine 2. yielded results qualitatively similar to those reported for T-ala; it appeared that the effect of fl-3-thienylalanine was less severe, both with respect to the inhibition of growth and the lability of the fl-galactosidase formed than was the effect of T-ala. However, no attempt was made to examine the effect of/5-3-thienylalanine incorporation in more detail. ACKNOWLEDGEMENTS
We should like to thank Professor K. Wallenfels of Freiburg University for a generous supply of crystalline E. coli, Strain ML 309 fl-galactosidase. We should like to thank Mrs. W. STERN and Mrs. P. ASHMAN for their excellent technical assistance. The investigations described here were supported by research grants from the National Science Foundation (G 15037) and the Public Health Service (A-5868). REFERENCES 1 G. N. COHEN AND F. GROS, Ann. Rev. Biochem., 29 (196o) 525 • i~ G. N. COHEN AND R. MUNIER, Biochim. Biophys. Acta, 31 (1959) 347. 8 R. L. MUNIER, Compt. Rend., 250 (196o) 3524. 4 M. H. RICHMOND, Biochem. J., 77 (196o) 121. s E. W. WESTHEAD AND P. D. BOYER, Biochim. Biophys. Acta, 54 (1961) 144. J. JANECEK AND H. V. RICKENBERG, Bacteriol. Proc., (1962) lO5. M. COHN AND J. MONOD, Biochim. Biophys. Acta, 7 (1951) 153. 8 H. V. RICKENBERG, C. YANOFSKY AND D. M. BONNER, J. Bacteriol., 66 (1953) 683. O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, ,[. Biol. Chem., 193 (I95 I) 265.
Biochim. Biophys. Acta, 8i (1964) lO8-12i
fl-2-THIENYLALANINEIN E.
coli fl-GALACTOSIDASE
12I
10 j . MANDELSTAM, Biochem. J., 69 (1958) IiO. n A. S. L. H u , R. G. WOLFE AND F. J. REITHEL, Arch. Biochem. Biophys., 81 (1959) 500. 12 H. G. KUNKEL AND R. J. SLATER, Proc. Soc. Exptl. Biol. Med., 80 (1952) 42. 13 B. ROTMAN AND S. SPIEGELMANN, J. Bacteriol, 68 (1954) 419 • 14 M. COHN AND A. M. TORRIANI, J. Immunol., 69 (1952) 471. is H. V. RICKENBERG, G. IxT. COHEN, G. BUTTIN AND J. IV[ONOD, Ann. Inst. Pasteur, 91 (1956) 829. is M. L. ZARNITZ, Dissertation, F r e i b u r g U n i v e r s i t y (Germa ny), 1958. 17 M. H. RICHMOND, Biochem, J., 77 (196o) 112. 18 j . ~q~. ANDERSON AND H. V. RICKENBERG, J. Bacteriol., 80 (196o) 297. 19 K. WALLENFELS AND A. ARENS, Biochem. Z., 332 (196o) 247. 20 R. MUNIER AND G. N. COHEN, Biochim. Biophys. Acta, 31 (1959) 378. 2x D. KERRIDGE, Biochim. Biophys. Acta, 31 (1959) 579. ~2 R. L. MUNIER AND G. SARRAZIN, Compt. Rend., 254 (1962) 2853.
Biochim. Biophys. Acta, 81 (i964) i o 8 - i 2 i