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Protein synthesis in Bacillus stearothermophilus
BIOCHIMICA ET BIOPHYSICA ACTA
376
BBA 95465
P R O T E I N S Y N T H E S I S IN BACILLUS S T E A R O T H E R M O P H I L U S B. B U B E I . A AND E. S. H O L D S W O R T H "
Biochemistry Department, University o/ Adelaide, Adelaide, Australia) (Received D e c e m b e r 23rd, I965)
SUMMARY
When 14C-labelled amino acids were incorporated by protoplasts from Bacillus stearothermophilus, the highest specific activity of protein appeared in the cytoplasmic membranes. The 2o ooo ×g supernatant from a preparation of lysed cells contained enzymes capable of activating amino acids and transferring them to tRNA. This system, lost 50 o,,~oof its activity~ on heating at 6o ° for IO min. The isolated aminoa c i d - t R N A complex was also heat labile. The 2o o o o x g supernatant had only a limited ability to incorporate amino acids into protein even when supplemented with artificial m R N A (poly-U). The most active site for incorporation in broken protoplasts was the cytoplasmic membranes. Membranes had an optimum temperature for incorporation of 65 ° and the membranes lost only 20 ° o of their activity for incorporating amino acids into protein on heating at 65 ° for 2o min. Factors contributing to the ability of B. stearothermophilus t,) grow at 6o ° are discussed.
INTR()DUCTION
Tile previous paper I showed that turnover of protein and nucleic acids in
Bacillus stearothermophilus is very rapid and that rapid repair of (:ell substance might allow this organism to withstand the damage brought about by high temperatures. Since protein and nucleic acid synthesis is brought about by enzTyanes and ribonucleoprotein particles it is of interest to find out whether the mechanisms for synthesis are heat stable. This paper examines some aspects of protein synthesis and suggests that heat stability depends on a high degree of intracellular organisation.
METHODS
The method of growing and harvesting B. stearothermophilus, preparation of protein and nucleic: acid for counting and the estimation of radioactivity are described in the previous paper a. A b b r e v i a t i o n : poly-U, polyuridylic acid. • P r e s e n t address: B i o c h e m i s t r y D e p a r t m e n t , Medical School, U n i v e r s i t y of T a s m a n i a , H o b a r t , Australia.
Biochim. Biophys. Acta, i23 (~966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
377
Lysis o/ B. stearothermophilus IO g wet weight of cells were resuspended in o.13 M Tris-HC1 (pH 7.5) to a volume of 42 ml and made 0.02 M MgC12 and 0.05 M NaC1. When protoplasts were needed as such for study, the buffer was also made IO % (w/v) sucrose. Lysozyme, 50 mg, dissolved in 8 ml o.13 M Tris-HC1 (pH 7.5) was added and the mixture incubated 15 min at 37 °. A microscopic check at this stage showed that cells had become protoplasts. The suspension was cooled in ice and centrifuged at 5 o o o x g for 15 rain. If the protoplasts were to be used as such they were washed once with o.13 M Tris-HCl (pH 7.5) containing lO % (w/v) sucrose and then resuspended in this buffer. For the further lysis of the protoplasts two methods were used: (i) A paste of the protoplasts was poured into IO vol. of ice-cold o.13 M Tris-HCl (pH 7.5). Lysis was complete in 30 min at o °. (ii) The protoplasts (20 ml) were treated with 20 mg perfluorooctanoic acid in 2 ml o.13 M Tris-HCl (pH 7.5). The protoplasts disintegrated within 5 min at o °. The volume of the lysed preparations was adjusted to suit the experimental conditions.
Preparation o~ cell lractions (a) The crude lysate was centrifuged at the following speeds, 5 o o o x g for 3o min, 20 ooo x g for 60 min, lO5 ooo x g for 60 min and the various pellets were collected and the Io 5 ooo x g supernatant preserved. (b) For the preparation of membranes the 5 o o o x g pellet from the lysate prepared by osmotic shock (as (i) above) was resuspended in Io % (w/v) sucrose in o.13 M Tris-HC1 (pH 7-5) and layered on top of a discontinuous sucrose gradient of IO, 20, 30, and 40 o,~ (w/v). The material was centrifuged at 15oo x g for 90 min. The membranes accumulated as a distinct fraction at the upper part of the 30 % (w/v) sucrose layer. The membranes were removed and collected by centrifugation at 5000 x g for 15 min. For some purposes the gradient centrifugation was repeated twice more. (c) Ribosomes were prepared from the lysate made by osmotic shock (method (i) above) and adjusted to I mM MgCl~ final concentration. It was centrifuged twice for 60 rain at 20 ooo x g to remove cell debris. The supernatant was then centrifuged at lO 5 ooo x g for 90 min. The pellet of ribosomes was resuspended in o.13 M TrisHCI (pH 7.5) and containing I mM MgCl v (d) pH-5 fraction was prepared from the lO5 ooo × g supernatation after removal of the ribosomes, by adjusting to pH 5 with I M acetic acid. The procedure was done at o ° and after standing for 5 min the precipitate was collected by centrifugation at lO5 ooo x g for 3 ° min. The pH- 5 fraction was dissolved in o.13 M Tris-HCl (pH 7.5).
Preparation o/ 14C-labelled amino acid-tRNA complex The pH-5 fraction equivalent to 115 mg protein and 12.5 mg nucleic acid were incubated in 15 ml o.13 M Tris-HC1 (pH 7.5) containing 15o ffmoles A T P (potassium salt), I5offmoles MgCl~ and 5offC 14C-labelled Chlorella hydrolysate (IOoffC/mg). After IO min at 60 ° the mixture was cooled in ice, mixed with an equal volume of 80 % (w/v) phenol in water and shaken vigorously. The aqueous phase was separated,
B~ochim. Biophys. Acta, 123 (1966) 376--389
378
B. BUBELA, ]g. S. HOI.I)SWORTH
extracted twice with ether, then made 2 % with respect to potassium acetate and the RNA precipitated with 2 vol. cold methanol. The preparation was left overnight at ....2o ° and the precipitate collected. The precipitated complex was dissolved in water and dialysed against o.13 M Tris--HCl (pH 7.5). The yield was 5 mg R N A amino acid complex with a specific activity of 420 mpC.'mg.
Preparation o / R N A /rom B. stearothermophilus What was required was mRNA but since this has a rapid turnover 1 no satisfactory way could be devised of obtaining this material separate from the other nucleic acids present in the bacteria. Cells obtained by harvesting B. stearothermophilus during logarithmic growth were suspended in water (Ae00,,,~,--IO), treated with an equal volume of phenol and the RNA separated as described above.
Analytical methods Protein was estimated by a modification of the method of ~VESILEY AND LAMBETtl2. The orcinol method of DISCHEa was used to estimate RNA.
Measurement o/amino acid activation (a) The method of formation of amino acid hydroxamates by pH- 5 fraction was essentially that described by HOAGLAND, KELLER AND ZAMECNIK4. (b) The incorporation of 14C-labelled amino acids from Chlorella hydrolysate into the pH- 5 fraction was performed as described by FRASER AND HOLI)SWORTII5. The radioactivity associated with nucleic acid and protein that was precipitated by ice-cold 5 o, ,,o trichloroacetic acid was assumed to be a measure of amino acid activation.
Incorporation o~ amino acids into whole cells and protoplasts The conditions used were those described in the previous paper 1 except that when protoplasts were used the medium contained in addition IO °:o (w/'v) sucrose. When subcellulm fractions were prepared the incubation mixture was cooled in ice and the cells lysed as described.
Incorporation o/ amino acids into subcellular /ractions (a) The supernatant obtained at 2o ooo ×g (2.5 mg protein) was incubated with Io/,moles ATP, o.2 5/,mole GTP, 5/,moles phosphoenolpyruvate, 2opmoles MgC12, ISo/,moles KCI, 5o/*g casein hydrolysate, I/,C of 14C-labelled Chlorella hydrolysate (or [14Cjphenylalanine) in 3 ml o.i 3 M Tris-HC1 (pH 7.5)- The reaction was stopped by chilling in ice and addition of an equal volume of ice-cold IO % trichloroacetic acid. The materials were prepared for counting the radioactivity as previously describedL (b) Washed membranes, 15o-25o/~g protein, were incubated in I ml of the following medium: 2o/~moles KC1, 5o/,moles Tris, 5° pmoles maleic acid, 5 !,moles MnCl,,, I/1mole MgCI2, 2/,moles ATP, o.1/~mole of each GTP, UTP, CTP, dd\TP,.
Biochim. Biophys. Acta, ~23 (I906) 370-389
PROTEIN SYNTHESIS IN A THERMOPHILE
379
dGTP, dTTP and dCTP, 2 mg casein hydrolysate and I FC of Chlorella hydrolysate (or [14C]phenylalanine). After incubation for a given time at the experimental temperature the mixture was chilled in ice. From this point different procedures were employed. In some experiments the radioactivities incorporated into material stable to cold and hot trichloroacetic acid were measured 1. To determine the distribution of radioactivity within the membrane the incubation mixture was centrifuged at 20 o o o × g for 15 min and the membranes were resuspended in water and divided into two equal portions. The 20 o o o × g supernatant was treated with 5 °o trichloroacetic acid to yield any protein that had leached from the membranes. One portion of the membranes was mixed with an equal volume of 8o o/o (w,'v) phenol in water and the amino acid-tRNA complex prepared as described above. The second portion was treated successively with 9 vol. acetone, methanol (twice), chloroform and ether to give a lipid extract that was evaporated under a stream of N2. The lipid was dissolved in I ml ethanol and 5 ml petroleum ether and the organic layer washed three times with 2 ml water. The residue of protein left after lipid extraction was heated in a boiling-water bath with 2 ml I M NaCI. The saline extract was treated with 2 vol. ethanol and the precipitated RNA-amino acid complex collected. The residue from the saline extraction was treated with hot trichloroacetic acid and solvent as previously described I and the protein counted for radioactivity.
Heat stability o/membrane-activating enzymes A soluble extract was made from washed membranes by treatment in o.13 M Tris-HC1 (pH 7-5) containing I mM perfluorooctanoate with ultrasonic vibration for IO min (6o-W M.S.E. ultrasonic disintegrator) the membranes being immersed in ice during treatment. The material was then centrifuged at lO5 ooo ×g for 30 rain. The supernatant and the original membranes were compared for their heat lability by heating them for tile specified times at 60 °, then estimating the residual amino acid-activating activity by the incorporation of 14C-labelled amino acids into material precipitated by ice-cold trichloroacetic acid (as described above).
RESULTS
Studies with whole cells and protoplasts Whole cells were incubated with x4C-labelled Chlorella hydrolysate for 2 and IO min, the cells lysed as described and the distribution of radioactivity in the cell fragments determined. Table I shows that material sedimenting at 5000 ×g haci the greatest specific activity. As the time of incubation increased a significant amount of radioactivity appeared in the lO5 ooo ×g pellet and supernatant and some of this was stable to hot trichloroacetic acid, i.e. represents protein synthesis. The 5000 ×g pellet, which contained the most radioactivity, was examined further by distribution on a sucrose gradient. The pellet gave two main fractions. /o sucrose layer appeared to be cell One which collected in the upper part of the 30 o~ membranes and fragments of cell wall when examined in the electron microscope. Biochim. Biophys. Acta, x23 (1966) 376-389
380
B. BUBELA, E. S. HOLDSWORTH
TABLE I DISTRIBUTION OF IIC-LABELLED AMINO ACIDS IN CELL F R A C T I O N S
The i n c u b a t i o n m i x t u r e contained 2o ml of bacterial s u s p e n s i o n (A700m# = IO) in o.13 M T r i s HC1 (pH 7.5), 2o/zC of 14C-labelled a m i n o acids (Chlorella hydrolysate, ioo #C/mg), 2oo mg of glucose, 2o m g of casein h y d r o l y s a t e in a t o t a l 4 ° ml of o.13 M Tris-HC1 (pH 7.5). The material w a s i n c u b a t e d for io m i n at 63 °, cooled to 37 ° and lysed as described in t h e METHODS section. The p r o t o p l a s t s were disrupted b y osmotic shock and the resulting material w a s separated into individual fractions. The r a d i o a c t i v i t y was m e a s u r e d and expressed as #/~C/mg of m a t e r i a l insoluble in h o t a n d / o r cold trichloroacetic acid.
Fraction
2 rain
Whole cells 5ooo × g pellet 20 ooo × g pellet IOO ooo × g pellet IOO ooo × g s u p e r n a t a n t
IO rain
Cold trichloroacetic acid
Hot trichloroacetic acid
Cold trichloroacetic acid
Hot trichloroacetic acid
26o 445 65 8o 25
I9o 298 45 5° io
78o 133o 195 278 402
58o 93 ° 137 21o 28o
A smaller fraction which collected at the bottom of the 5o % sucrose layer had the highest specific activity and was rich in nucleic acid. The radioactivity associated with this heavy fragment was labile to treatment with hot trichloroacetic acid. Since the distribution of radioactivity in the lysed cell could be complicated by the presence of fragments of cell wall, experiments were made with protoplasts. After incorporation of amino acids and lysis of the protoplasts by osmotic shock, the subcellular fractions were prepared and the 5000 ×g pellet was further purified by distribution on a sucrose gradient as described for the preparation of
TABLE II MEMBRANE
PARTICIPATION
IN PROTEIN
SYNTHESIS
P r o t o p l a s t s p r e p a r e d as in the METHODS section were incubated in the presence of 1*C-labelled a m i n o acids at 63 °. The i n c u b a t i o n m i x t u r e contained I m l o f a suspension of p r o t o p l a s t s (A700m# = IO) in o.13 M Tris-HC1 (pH 7.5), i #C of 14C-labelled a m i n o acids (Chlorella hydrolysate, I o o # C / rag), IO m g of glucose, I m g o f casein h y d r o l y s a t e in t o t a l v o l u m e of 2 ml of IO % (w/v) sucrose in o.13 M Tris-HC1 (pH 7.5). One sample w a s i n c u b a t e d for 2o sec at 63 °, a second s a m p l e for 3 m i n and a t h i r d sample for 3 min, t h e n 3 mg of casein h y d r o l y s a t e were added and the reaction m i x t u r e i n c u b a t e d for a f u r t h e r 3 min. After the incubation, 5/*g of chloramphenicol were added to each sample, which w a s frozen immediately in a solid CO2-acetone freezing mixture. The p r o t o p l a s t s were disrupted b y adding the frozen material to 6 vol. of ice-cold water. The individual fractions were p r e p a r e d and the radioactivity in h o t a n d / o r cold trichloroacetic acid-insoluble material w a s measured. S y m b o l s N A and P represent nucleic acids and proteins, respectively.
Material
5000 × g pellet Membranes io ooo x g pellet 135 ooo × g pellet I35 ooo × g s u p e r n a t a n t
2o sec (minGling)
3 rain (mt~C /mg )
3 + 3 rain (ml~C /mg )
NA
P
NA
P
NA
P
o.612 o.712 O.lO 3 0.250 o.o96
0.298 0.385 o.o96 o. 112 o.121
1.o3o 1.65o o.2o 7 o.416 O.lO 7
o.5ol 0.652 o.125 o.2ol o.216
0.065 0.976 o.165 0.375 o.iio
0.572 o.765 o.137 o.135 1.o7o
Biochim. Biophys. Mcta, 123 (1966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
38I
membranes. From Table II it can be seen that the membrane fraction contained the greatest specific activity except where the radioactivity was "chased" into the lO5 ooo × g supernatant by further incubation with non-labelled amino acids. The material in the membrane fraction did in fact appear to be protoplast membranes as can be seen from the electron micrograph (Fig. i). In the 2o-sec incubation it appears that the membrane fraction was the first to become radioactive.
Incorporation o/ amino acids into subcellular /ractions Activation o/ amino acids. The formation of amino acid hydroxamates was taken as a measure of amino acid activation. Control tubes in which there were no added amino acids served to measure the small amount of hydroxamate formation from endogenous amino and fatty acids. Amino acid-activating systems are precipitated in the pH-5 fraction in mammalian systems ~ and it was interesting to find that the system in B. stearothermophilus could be precipitated in the same manner.
Fig. I. Electron micrographof protoplast membrane of B. stearothermophilus. method
Prepared by the
of ~ELLENBERG, RYTER AND SEEHAND 6.
Biochim. Biophys. Acta, 123 (1966) 376-389
382
B. BUBEI.A, E. S. HOI.I)SWORTtI
As an assurance t h a t a m i n o acid a c t i v a t i o n was being measured, e x p e r i m e n t s were m a d e to show t h a t the p H - 5 fraction could i n c o r p o r a t e ~4('-labelled a m i n o acids into m a t e r i a l p r e c i p i t a t e d b y ice-cold trichloroacetic acid. This m e t h o d includes a further s t e p after a c t i v a t i o n i.e. a t t a c h m e n t of the a m i n o acids to t R N A . The results of the two m e t h o d s are v e r y similar ;is shown in Fig. 2. A c t i v a t i o n was not r a p i d until 35 -4o° h a d been reached and rose to a m a x i m u m at 59 61°. I t was observed t h a t i n c u b a t i o n of the p H - 5 fraction above 45 ~ caused a p r e c i p i t a t e to form. "1"o s t u d y the t h e r m a l s t a b i l i t y of the amino a c i d - a c t i v a t i n g enzymes the pH-5 fraction was h e a t e d for IO min at various t e m p e r a t u r e s and then tested for its a c t i v a t i n g a c t i v i t y b y the h y d r o x a m a t e m e t h o d . F r o m the results shown in Fig. 3 it can be seen t h a t the enzymes are not stable a b o v e 4 o°. The p r e c i p i t a t e s t h a t formed were inactive when tested separately. The concentration of n u c M c acid in solution did not change during the incubation.
~
,°I
a"0 8
30
2o~
¢
0.4-~
@-
0
I0
20
30
40
50
60
Temperature Fig. 2. Effect on t e m p e r a t u r e on a m i n o acid a c t i v a t i o n by pH- 5 fraction. The a m o u n t of a m i n o acid h y d r o x a n , a t e ( O ) o r t h e a m o u n t of t4('-labelled a m i n o acid t R N A c o m p l e x ( O ) formed in xo rain a t d i f f e r e n t t e m p e r a t u r e s was d e t e r m i n e d as de s c ri be d in t h e .METtlOI)S section.
2.5
°°t o6t o
20
o4
rl
1.0
0.2
20
30 40 50 Temperature
60
0.5
Fig. 3. S t a b i l i t y of the a m i n o a c i d - a c t i v a t i n g e n z y m e s to he a t . pH - 5 e n z y m e w a s h e a t e d for I o m i n a t t h e specified t e m p e r a t u r e s and c e n t r i f u g e d . The a m i n o a c i d - a c t i v a t i n g activity, rem a i n i n g in s o l u t i o n was m e a s u r e d by the h y d r o x a m a t e m e t h o d ((3). P r o t e i n r e m a i n i n g in s o l u t i o n ( 0 ) was m e a s u r e d as d e s c r i b e d in tile METIIOIJS section.
Biochim. Biophys. A cta, r23 (t,466) 376-389
383
PROTEIN SYNTHIt'SIS IN A THERMOPHILE
Thermostability o/the amino acid-tRNA complex. The ~4C-labelled a m i n o a c i d t R N A complex was prepared a n d dissolved in o.13 M T r i s - H C l (pH 7-5) a n d heated at 37 ° a n d 63 ° for specified times. The r a d i o a c t i v i t y in material t h a t could be precipitated with ice-cold trichloroacetic acid was regarded as stable complex. Fig. 4 shows the results obtained. Approx. 5 ° % of the complex was decomposed in io min at 63 ° . Incorporation o/amino acids into 2o ooo × g supernatant. Lysed cells prepared b y using perfluorooctanoic acid were used to prepare a 2 o o o o × g s u p e r n a t a n t . I n c o r p o r a t i o n of ~4C-labelled a m i n o acid into this s u p e r n a t a n t , subfractions prepared from it and the reconstituted system are shown in Table 1lI. The results show t h a t incorporation at o ° is relatively high or alternatively, the increase in going from o ° to 63 ~ was only' two-fold. Chloramphenicol did not alter the a m o u n t of incorporation at o ° but completely inhil)ited the difference between o ° a n d ()3 °. Almost all of the limited ability to incorporate a m i n o acids was in the material t h a t precipitates at pH 5. Trans/cr o/amino acids/tom the tRNA complex into proteins. Since the fraction t h a t precipitates at pH 5 appeared to incorporate amino acids an a t t e m p t was made to see whether amino acid could be transferred from the 14C-labelled a m i n o a c i d t R N A complex (prepared as described in the .~fETHODS section) into hot trichloroacetic acid-stable material. Fig. 5 shows t h a t the transfer takes place rapidly at 63 °. Stvdy o/ ribosomes. A s t u d y of the ribosomes of B. stearothermophilus was made at two c o n c e n t r a t i o n s of Mg"'. The s e d i m e n t a t i o n values in o.I M NaCl plus o.oi M MgC12 were 31 S, 50 S, and 73 S, a n d in o.I M NaCI plus I mM MgC12 these values were 28 S, 48 S, a n d 65 S. The m a j o r portions were in the 73-S and 65-S fractions. No particles were seen t h a t could correspond to polysomes. 100. ~
~ t - ~ -
0
.
~ U
60-
~" 0.6'
,~ 40.
o ~0.4
c
0 Q.
~0.2 c
0
5 10 20 30 T~me (m~n)
40 Tirne (rain)
Fig. 4. Effect of heat on amino acid tRNA complex, liC-labelled amino acid-tRNA complex was prepared as described in the METHODSsection and heated at .37° (S) and (~3° (O) for the specified times. The radioactivity precipitable with cold trichloroacetic acid is a measure of unchanged complex and was expressed as per cent of the original complex. Fig. 5. Transfer of t4C-labelled amino acids from the tRNA complex to proteins, t4C-labelled amino acid-tRNA complex (prepared as in the METHODSsection) was incubated for io rain at 4°° (0) or 63 ° (O) in the presence of pH-5 fraction. 14C incorporated into material stable to hot trichloroacetic acid was regarded as protein.
Biochim. t3iophys. Acta, Iz3 (J966) 376-389
B. BUBELA, E. S. HOLDSWORTH
384 TABLE III INCORPORATION
OF AMINO
ACIDS
INTO
2 0 OOO X g S U P E R N A T A N T ,
ITS
SUBFRACTIONS
AND
A RE-
C O N S T I T U T E D S Y S T E M , A T O °, 3 7 ° A N D 6 3 °
The i n c u b a t i o n m i x t u r e contained 20 ooo × g s u p e r n a t a n t (2.5 mg of protein), io #moles of ATP, o.25 # m o l e of GTP, 5/*moles of p h o s p h o e n o l p y r u v a t e , 5 °/~g of casein hydrolysate, i/~C of llC-labelled anlino acids (Chlorella hydrolysate, i o o / , C / m g ) , 21/~moles of MgC12, i8o/~moles of KCI in 3 ml o.13 M Tris-HC1 (pH 7.5). W h e r e individual subfractions were investigated, separately or in a mixture, the a m o u n t p r e s e n t was similar to the original c o m p o s i t i o n of the 2o ooo × g s u p e r n a t a n t . The material was i n c u b a t e d for io rain at the e x p e r i m e n t a l t e m p e r a t u r e . The radioactivity was m e a s u r e d and expressed as /HzC/mg of material insoluble in h o t trichloroacetic acid.
Material
rng per fraction
o°
37 °
63 °
20 ooo X g s u p e r n a t a n t Ribosomes p H - 5 fractions pH- 5 supernatant R e c o n s t i t u t e d material
2.5 ° 0.05 2. io 0.35 2. 5
15.5 9.2 9.7 12.3 13.6
18. 7 9-5 12.5 12. 7 16.6
30.0 9.5 24.7 15.o 23.7
Role o/poly-U in protein synthesis in B. stearothermophilus. Since there seemed to be no polysomes as such in the 20 ooo × g supernatant, experiments were made to study the effect of poly-U on incorporation of phenylalanine. Table IV shows that poly-U stimulates the incorporation of phenylalanine into peptide bonds and the results also suggest that there is some destruction of poly-U during the IO rain at 63 ° since if the material was added in portions a greater incorporation was obtained. It was again noticed that during incubation a precipitate formed presumably due to denaturation of protein. Since the ionic strength of the buffer used in these experiments is high, o.13, the experiment was repeated in buffer with ionic strength o.o13 and the incorporation studied at various temperatures. Table V shows that optimum incorporation took place at 45 ° and again a precipitate formed at temperatures above 45 °. Cell-free extracts prepared by grinding protoplasts with alumina 7 gave very similar results. T A B L E IV INFLUENCE
OF POLY-UON
THE INCORPORATION OF PHENYLALANINE
I N T O 2 0 OOO N g S U P E R N A T A N T
The incorporating m i x t u r e contained 20 ooo × g of s u p e r n a t a n t (3 mg of protein), IO/~moles of ATP, 0.25/~mole of GTP, 5 # m o l e s of p h o s p h o e n o l p y r u v a t e , io/~g of casein hydrolysate, i #C of uniformly labelled phenylalanine (specific activity i o o # C / m g ) , i o # g of phenylalanine, 21 /~moles of MgCI~, 18o/~moles of KC1 in 3 ml o. 13 M Tris HC1 (pH 7.5). To one aliquot of the prepa r a t i o n i n c u b a t e d at 63 °, lOO/~g of poly-U were added. To a n o t h e r aliquot 25/~g of poly-U were added every 2.5 rain during t h e io min of incubation. The radioactivity of the material w a s m e a s u r e d and expressed a s / ~ # C / m g of material insoluble in h o t trichloroacetic acid,
Temperature
Radioactivity
Increase above o ° incorporation
o° 63 ° 63 ° + poly-U 6 3 ° + p o l y - U added in p a r t s
16. 7 35-3 42.5 50.2
-2. I-fold 2.5-fold 3.o-fold
Biochim. Biophys. Acta, 123 (1966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
385
TABLE V INFLUENCE OF POLY-~J ON THE INCORPORATION AT DIFFERENT TEMPERATURES FOR IO MIN
OF PHENYLALANINE
I N T O 2 0 OOO X g S I J P E R N A T A N T
M e t h o d as for T a b l e I V w i t h t h e e x c e p t i o n t h a t t h e s t r e n g t h of t h e T r i s - H C 1 buffer w a s O.Ol 3 M. Po ly-U, 25 pg, was a d d e d a t 2.5-min i n t e r v a l s .
Temperature
Radioactivity (IHzC/mg )
o° 1°0 20 ° 3 °0 35 ° 4 °0 45 ° 5° 55 ° 60 °
28. 3 29.7 31.9 37.9 56. 7 75.4 98.6 81.8 73.3 60.2
1.0
/
..EEO.S o ~EO.6
g o
~o
?
0.4
£0.2 ~,
~b203o
,~o
50
40
Ternperoture
Fig. 6. I n c o r p o r a t i o n of a m i n o acids i n t o m e m b r a n e s . M e m b r a n e s p r e p a r e d from p r o t o p l a s t s of B. stearothermophilus were i n c u b a t e d w i t h 14C-labelled C hl ore l l a h y d r o l y s a t e as d e s c r i b e d in t h e METHODS section. The r a d i o a c t i v i t y i n c o r p o r a t e d i n t o m a t e r i a l s t a b l e t o cold ( O ) or h o t ( 0 ) t r i c h l o r o a e e t i c acid w a s m e a s u r e d a n d e x p r e s s e d as m/zC/mg of nucleic a c i d or p r o t e i n , r e s p e c t i v e l y .
1.0-
~
0.8 g .~0.6 ~0.4
o.2
10
20 30 40 Temperature
50
60
Fig. 7, I n c o r p o r a t i o n of a m i n o a c i d s i n t o m e m b r a n e s i n t h e p r e s e n c e of m R N A . E x p e r i m e n t a l : o n d i t i o n s as for Fig. 6 w i t h t h e i n c l u s i o n of m R N A i n t h e i n c u b a t i o n m i x t u r e . T h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o m a t e r i a l s t a b l e to cold ( O ) or h o t ( O ) t r i c h l o r o a c e t i c a c i d w a s m e a s u r e d a n d ~xpressed as mffC/mg of nucleic a c i d or p r o t e i n , r e s p e c t i v e l y .
Biochim. Biophys. Acta, 123 (1966) 376-389
386
B.
BUBELA,
E.
S. H O L D S W O R T H
Membranes as a site o~ protein synthesis It was reported b y S P I E G E L M A N 9 that isolated membranes from Escherichia coli incorporate amino acids into hot trichloroacetic acid-insoluble material. When membranes from B. stearothermophilus were incubated as described by SPIEGELMAN9 the results obtained in Fig. 6 were obtained. The 14C-labelled amino acids became attached mainly to nucleic acids, i.e. material unstable to hot trichloroacetic acid and there was only a small incorporation into protein. If the incubation mixture was supplemented with RNA extracted from B. stearothermophilus the situation was changed (Fig. 7) and amino acids became incorporated into protein. The optimum temperature for these incorporations was 65 ° , although the membranes tended to flocculate at temperatures above 45 ° . The incorporation of amino acid into membranes was studied in more detail b y following the distribution of radioactivity in lipids, attached to nucleic acid, or incorporated into protein, as affected by time of incubation. It is apparent that the highest specific activity appears firstly in the lipid fraction (Table VI), then in the amino acid-tRNA complex and finally in the membrane protein. Some radioactivity protein also appeared in free solution in the incubation mixture. Repeated extraction of the lipo-amino-acid complex in petroleum ether solution with I o/~} (w/v) aqueous solution of casein hydrolysate did not remove the radioactivity into the aqueous phase. The lipo-amino-acids were examined by chromatography on W h a t m a n 3 MM paper using benzene-n-butanol (I : I, v/v) saturated with water as a solvent. The lipo-amino-acids had R F ' s between 0. 4 and 0. 7 in this solvent whereas the free amino acids had RF's below o.15. TABLE
VI
INCORPORATION
OF AMINO
ACIDS
INTO
MtgMBRANES
AT 6 0 ~:
M e m b r a n e s w e r e i n c u b a t e d f o r 3 ° sec, I r a i n a n d 3 r a i n i n t h e m e d i u m d e s c r i b e d f o r F i g . 7. T h e v a r i o u s f r a c t i o n s w e r e p r e p a r e d a s d e s c r i b e d i n t h e METHODS s e c t i o n . T h e r a d i o a c t i v i t y is e x p r e s s e d a s m # C / m g o f R N A .
Fraction RNA-amino acid (phenol method) membrane RNA amino acid Lipo amino acid Membrane protein Supernatant protein
3 o sec
i rain
3 rain
7.8
lO. 5
13.2
-15. 3 i.i --
-i8.i 2.6 --
15. 3 "23.3 lO.2 7- i
Thermostability o/ the activating enzymes o~ membranes The incorporation of amino acids into protein by the membranes was found to decrease only 20 % after heating for IO rain at 63 ° (ref. IO). A comparison was made of the heat stability of the amino acid-activating system in membranes with a soluble activating system prepared from the membranes. The results shown in Fig. 8 indicate that the soluble activating system prepared from the membranes was less stable to heat than that present in the original membranes. No significance can be attached to the difference in specific activity between the two preparations since they had different proportions of protein to RNA. Biochim. B i o p h y s . Acta, 123 ( 1 9 6 6 ) 3 7 6 - 3 8 9
PROTEIN SYNTHESIS IN A THERMOPHILE
387
looJ 90-
8o! 7e4 %60 50 40 3O
oi 2
4 6 Time (rain)
8
10
Fig. 8. H e a t s t a b i l i t y of a m i n o a c i d - a c t i v a t i n g s y s t e m s . M e m b r a n e s p r e p a r e d f r o m p r o t o p l a s t s of B. stearothermophilus ( 0 ) a n d a soluble p r e p a r a t i o n p r e p a r e d b y u l t r a s o n i c t r e a t m e n t of t h e m e m b r a n e s ( 0 ) were h e a t e d a t 63 ° for t h e specified times. T h e residual a m i n o a c i d - a c t i v a t i n g a c t i v i t y ( m e a s u r e d b y i n c o r p o r a t i o n of laC-labelled a m i n o acid into m a t e r i a l p r e c i p i t a t e d b y cold trichloroacetic acid, see t h e METHODS Section) w a s e x p r e s s e d as a p e r c e n t a g e of t h e original activity.
DISCUSSION
Whole cells of B. stearothermophilus have been shown to incorporate 14Clabelled amino acids into protein with an optimum temperature of 60 ° (ref. I). When cells prepared in this way were disintegrated and separated into fractions b y centrifugation, it was found that the highest specific activity was located in membranes (Table I and Fig. I). Protein synthesised b y the membranes m a y become part of the soluble cytoplasm since the radioactivity incorporated into membranes could be ,,chased" into the soluble part by further incubation with non-labelled amino acids (Table II). The supernatant obtained at 20 ooo ×g from lysed cells of B. stearothermophilus contained an amino acid-activating system that had an optimum activity of approx. 60 ° using an incubation of IO rain. It was curious to find that activation of amino acids was very limited until temperatures above 35 ° were used. In these experiments there is no question of transport of amino acid to the site of activation since the activating system was soluble. Calculation of the heat of activation from the data in Fig. 2 gave a figure of 12 ooo cal, which is similar to that calculated for the incorporation of amino acids ] . Therefore, this is another fragment of evidence that the activation of enzymes from this organism requires higher temperatures than enzymes from mesophilic bacteria. This aspect was discussed in the previous paper ]. A_bove 4 °° the activating system was heat labile and 5o %was destroyed b y h e a t i n g ro min at 6o °. That the amino acids were activated was shown by the isolation of an ~mino a c i d - t R N A complex. This complex heated in buffer (with absence of enzymes) was thermolabile. A similar observation was made on the isolated leucine-tRNA =omplex from B. stearothermophilus by ARCA et al. ]1. An unexpected finding was that pH-5 fraction prepared from a lO5 ooo ×g ;upernatant of lysed cells, therefore presumably free from ribosomes, could transfer 4C-labelled amino acids from their complex with t R N A to material stable to hot :richloroacetic acid. The pH-5 fraction in fact constituted the most active part of the ,'o ooo × g supernatant when this was investigated for its ability to incorporate amino tcids into protein. However, the ability of the 2o ooo ×g to incorporate amino acids Biochim. Biophys. Acta, 123 (1966) 376-389
388
B.t'~UBF.I.A, IL. S. HOI.DS',A'ORTH
into protein was v e r y low c o m p a r e d with t h a t of whole ce.lls or protoplasts. One reason for this m a y be t h a t we could find no polysomes in our p r e p a r a t i o n . The largest particles were ribosomes of 7o S. MANGIANTINI et al) 2 r e c e n t l y r e p o r t e d t h a t particles of ~oo-S t y p e only a p p e a r e d in e x t r a c t s from B. stearothermotbhilus in Tris buffer c o n t a i n i n g 5 mM m a g n e s i u m acetate. The work on t u r n o v e r of nucleic acids r e p o r t e d in the previous p a p e r ~ showed t h a t labile nucleic acid had a life of only I min. Therefore, the absence of polysomes was not unexpectecl. Even when the 2o ooo x g s u p e r n a t a n t was s u p p l e m e n t e d with poly-U the incorporation, although s t i m u l a t e d , was still v e r y small. FRIEI).~IAN AND \VI.;INSTEIX la also found poly-Us t i m u l a t e d p h e n y l a l a n i n e i n c o r p o r a t i o n in a cell-free e x t r a c t of /4. stearothermophilus. T h e o p t i m u m t e m p e r a t u r e for i n c o r p o r a t i o n of p h e n y l a l a n i n e in this system was 45 ° a n d in m a n y e x p e r i m e n t s it was noticed t h a t heating the 2o ooo × g s u p e r n a t a n t a b o v e 45 ° gave a p r e c i p i t a t e of protein. Thus it a p p e a r s t h a t the isolated cell c o n t e n t s arc'. u n s t a b l e to heat. Ribosomes from B. stearolhermophilus have recently been shown to be more stable to heat t h a n those from E. coli L2, a l t h o u g h the tests were of physical p r o p e r t i e s and not of their function in protein synthesis. The most i m p o r t a n t site of a m i n o acid i n c o r p o r a t i o n a p p e a r s to be in the m e m b r a n e s o b t a i n e d from p r o t o p l a s t s of /4. stearothermophilus. This agrees with the obs e r v a t i o n s m a d e with whole cells. Cell m e m b r a n e s t h a t h a d been washed three times with o.I3 1~1 Tris HCI (pH 7.5) a n d were p r e s u m a b l y free from soluble proteins, were able to a c t i v a t e a m i n o acids a n d transfer t h e m to m a t e r i a l stable to t r e a t m e n t with hot trichloroacetie acid, i.e. proteins. The m a j o r incorporation was into an amino acid--. R N A c o m p l e x w h i c h was e x t r a c t e d and shown to contain r a d i o a c t i v i t y . W h e n crude. m R N A was included in the i n c u b a t i o n m i x t u r e for the m e m b r a n e s , much more a m i n o acid was i n c o r p o r a t e d into protein. The. o p t i m u m t e m p e r a t u r e for incorporation into m e m b r a n e s was ~5 ~. In c o n t r a s t to the 2o o o o × g s u p e r n a t a n t there was a large i n c r e m e n t of i n c o r p o r a t i o n between o ~' and 6o °, also the a m o u n t of i n c o r p o r a t i o n o b t a i n e d when using m e m b r a n e s was comparalfie with t h a t o b t a i n e d with p r o t o p l a s t s . M e m b r a n e s could be h e a t e d for 2o min at 65 ° with the loss of only 2o
Biochi~z. IJiophys. Acta, 1, 3 (tc~o0)37~J-~S9
PROTEIN SYNTttESIS IN A THERMOPHILE
389
thesis, stated that there was no proof that they were obligatory intermediates in protein synthesis. The complexes may be transport forms of amino acids or a storage form of the activated amino acids. There ix a growing volume of evidence that the cytoplasmic membrane ix an important site of protein synthesis in bacteria e.t,. in 13. megateri.um 14, in t:.'. colig, le, in B. subtilis :~, in Streptococcus /aecalis 1~. That this is indeed protein synthesis is suggested by the observation 10 that a membrane preparation from E. coli would produce alkaline phosphatase. In B. stearothermophilus not only is the membrane the most active site for the incorporation of amino acids but the results indicate that the mechanism for protein synthesis is more stable to heat when it ix part of the membrane. Several observations in this and the accompanying paper 1 may have a bearing on the ability of 13. stearothermophilus to grow at high temperatures. Firstly the energy of activation of the enzymes in thermophiles may be higher than in mesophiles, although not enough enzymes have been studied. Secondly, the turnover of protein and nucleic acid 1 is more rapid in B. stearothermophilus than in E. coli thus more rapid repair of heat damage ix possible. Thirdly, it seems that organisati()n into membrane-type structures can confer a certain amount of heat stability on enzymes. Further observations on this aspect will be presented in a subsequent paper.
REFEI{EN('ES i 13. BUBELA A.ND E. S. HOLDSWORTJL Biockim. Biophys..4cta, I23 (r966) 364. 2 J. XYESTI.EY AND J. I.AMICE'rH, Biochim. Biophys. Acta, 4 ° ( I 9 6 o ) 364 . 3 Z. DISCItE, in E. CIIARGAFF AND J. N. I)AVIOSON, The Nucleic Acids, Vol. I, Academic Press, New York, 195.5 , p. 28.5. 4 ix{" B. HOAGLAND, E. 13. KELLER AND }?. (;. ZAMECNIK, .[. Biol. Chem., 218 (19.56) 34.5. 5 ix|- J. FRASER AND E. S. HOLDSWORTII, Nature, 183 (1959) 519 . 0 ]{ELLENI3ERG, RU'rER AND SEEIIAND, J . Biophys. Biochem. Cytol., 4 (I958) 67t" 7' M. B. I]OAGLAND, Btochim. Biopkys. Acta, 1~ (1955) 228. g iX[. ~V. NIRI.;NBERG AND J. H. iXIATTHAEI, Proc. Natl. Acad. Sci. U.N., 47 ( 1 9 6 I ) t588. 0 S. SPIILGELMAN, in G. "J['UNEVALL, Recent Progress in 3Itcrobiology, Ahnquist, \Viksell-Stockholm, I958, p. 81. io ]-$. I~UBELA, Some .4spects o/ Protein Synthesis in Tkermaphilic Bacteria, l~h. D. Thesis, Uni vcrsity of Adelaide, 19()4, p. I2o. 11 iX{. :\RCA, C. CALVORI, L. FRONTALLI AND (;. TECCE, Biochem. Biophys. Res. Com,,un., lo (I963) II 7. I2 M. T. MANGIANTINI, G. TECCI-;, G. TOS(_'HI AND A. TRENTALANCE, Biochim. Bioph)'s. :Iota, Io3 (~965) 252. 13 S. FRIEI)MAN AND I. ~,VIMNSTEIN, Federation Proc., 23 ( I 0 6 4 ) 164. x4 (;. l.). tlu.~'rER AND R. A. GOODSALL, Biochem. J., 78 (1901) .ej{'i,|. I 5 1¢.. \ ¥ . HENDLER, Biochim. Biopkys. Acta, 49 (1961) 297. 16 J. TANI AND }{. V}~. HENDLER, B*ochim. Biophys. ,4eta, 8o (1904) 279. 17 M..NORMURA, J. HOSODA AND S. NISHIMURA, Biockim. Biophys. Acta, 29 (I958) I 6 L x8 L. 1.). MOORE A.~'D \V. \V. UMBREIT, Biochim. Biopkys..4eta, IO3 (I965) 406. I0 D. H. L. BISHOP, I{. CHANTAL AND B..NIS.MAN, Biochem..]., 90 ( I 9 0 4 ) 378.
Biochim. Biophys. Acta, i23 (1966) 376-389
376
BBA 95465
P R O T E I N S Y N T H E S I S IN BACILLUS S T E A R O T H E R M O P H I L U S B. B U B E I . A AND E. S. H O L D S W O R T H "
Biochemistry Department, University o/ Adelaide, Adelaide, Australia) (Received D e c e m b e r 23rd, I965)
SUMMARY
When 14C-labelled amino acids were incorporated by protoplasts from Bacillus stearothermophilus, the highest specific activity of protein appeared in the cytoplasmic membranes. The 2o ooo ×g supernatant from a preparation of lysed cells contained enzymes capable of activating amino acids and transferring them to tRNA. This system, lost 50 o,,~oof its activity~ on heating at 6o ° for IO min. The isolated aminoa c i d - t R N A complex was also heat labile. The 2o o o o x g supernatant had only a limited ability to incorporate amino acids into protein even when supplemented with artificial m R N A (poly-U). The most active site for incorporation in broken protoplasts was the cytoplasmic membranes. Membranes had an optimum temperature for incorporation of 65 ° and the membranes lost only 20 ° o of their activity for incorporating amino acids into protein on heating at 65 ° for 2o min. Factors contributing to the ability of B. stearothermophilus t,) grow at 6o ° are discussed.
INTR()DUCTION
Tile previous paper I showed that turnover of protein and nucleic acids in
Bacillus stearothermophilus is very rapid and that rapid repair of (:ell substance might allow this organism to withstand the damage brought about by high temperatures. Since protein and nucleic acid synthesis is brought about by enzTyanes and ribonucleoprotein particles it is of interest to find out whether the mechanisms for synthesis are heat stable. This paper examines some aspects of protein synthesis and suggests that heat stability depends on a high degree of intracellular organisation.
METHODS
The method of growing and harvesting B. stearothermophilus, preparation of protein and nucleic: acid for counting and the estimation of radioactivity are described in the previous paper a. A b b r e v i a t i o n : poly-U, polyuridylic acid. • P r e s e n t address: B i o c h e m i s t r y D e p a r t m e n t , Medical School, U n i v e r s i t y of T a s m a n i a , H o b a r t , Australia.
Biochim. Biophys. Acta, i23 (~966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
377
Lysis o/ B. stearothermophilus IO g wet weight of cells were resuspended in o.13 M Tris-HC1 (pH 7.5) to a volume of 42 ml and made 0.02 M MgC12 and 0.05 M NaC1. When protoplasts were needed as such for study, the buffer was also made IO % (w/v) sucrose. Lysozyme, 50 mg, dissolved in 8 ml o.13 M Tris-HC1 (pH 7.5) was added and the mixture incubated 15 min at 37 °. A microscopic check at this stage showed that cells had become protoplasts. The suspension was cooled in ice and centrifuged at 5 o o o x g for 15 rain. If the protoplasts were to be used as such they were washed once with o.13 M Tris-HCl (pH 7.5) containing lO % (w/v) sucrose and then resuspended in this buffer. For the further lysis of the protoplasts two methods were used: (i) A paste of the protoplasts was poured into IO vol. of ice-cold o.13 M Tris-HCl (pH 7.5). Lysis was complete in 30 min at o °. (ii) The protoplasts (20 ml) were treated with 20 mg perfluorooctanoic acid in 2 ml o.13 M Tris-HCl (pH 7.5). The protoplasts disintegrated within 5 min at o °. The volume of the lysed preparations was adjusted to suit the experimental conditions.
Preparation o~ cell lractions (a) The crude lysate was centrifuged at the following speeds, 5 o o o x g for 3o min, 20 ooo x g for 60 min, lO5 ooo x g for 60 min and the various pellets were collected and the Io 5 ooo x g supernatant preserved. (b) For the preparation of membranes the 5 o o o x g pellet from the lysate prepared by osmotic shock (as (i) above) was resuspended in Io % (w/v) sucrose in o.13 M Tris-HC1 (pH 7-5) and layered on top of a discontinuous sucrose gradient of IO, 20, 30, and 40 o,~ (w/v). The material was centrifuged at 15oo x g for 90 min. The membranes accumulated as a distinct fraction at the upper part of the 30 % (w/v) sucrose layer. The membranes were removed and collected by centrifugation at 5000 x g for 15 min. For some purposes the gradient centrifugation was repeated twice more. (c) Ribosomes were prepared from the lysate made by osmotic shock (method (i) above) and adjusted to I mM MgCl~ final concentration. It was centrifuged twice for 60 rain at 20 ooo x g to remove cell debris. The supernatant was then centrifuged at lO 5 ooo x g for 90 min. The pellet of ribosomes was resuspended in o.13 M TrisHCI (pH 7.5) and containing I mM MgCl v (d) pH-5 fraction was prepared from the lO5 ooo × g supernatation after removal of the ribosomes, by adjusting to pH 5 with I M acetic acid. The procedure was done at o ° and after standing for 5 min the precipitate was collected by centrifugation at lO5 ooo x g for 3 ° min. The pH- 5 fraction was dissolved in o.13 M Tris-HCl (pH 7.5).
Preparation o/ 14C-labelled amino acid-tRNA complex The pH-5 fraction equivalent to 115 mg protein and 12.5 mg nucleic acid were incubated in 15 ml o.13 M Tris-HC1 (pH 7.5) containing 15o ffmoles A T P (potassium salt), I5offmoles MgCl~ and 5offC 14C-labelled Chlorella hydrolysate (IOoffC/mg). After IO min at 60 ° the mixture was cooled in ice, mixed with an equal volume of 80 % (w/v) phenol in water and shaken vigorously. The aqueous phase was separated,
B~ochim. Biophys. Acta, 123 (1966) 376--389
378
B. BUBELA, ]g. S. HOI.I)SWORTH
extracted twice with ether, then made 2 % with respect to potassium acetate and the RNA precipitated with 2 vol. cold methanol. The preparation was left overnight at ....2o ° and the precipitate collected. The precipitated complex was dissolved in water and dialysed against o.13 M Tris--HCl (pH 7.5). The yield was 5 mg R N A amino acid complex with a specific activity of 420 mpC.'mg.
Preparation o / R N A /rom B. stearothermophilus What was required was mRNA but since this has a rapid turnover 1 no satisfactory way could be devised of obtaining this material separate from the other nucleic acids present in the bacteria. Cells obtained by harvesting B. stearothermophilus during logarithmic growth were suspended in water (Ae00,,,~,--IO), treated with an equal volume of phenol and the RNA separated as described above.
Analytical methods Protein was estimated by a modification of the method of ~VESILEY AND LAMBETtl2. The orcinol method of DISCHEa was used to estimate RNA.
Measurement o/amino acid activation (a) The method of formation of amino acid hydroxamates by pH- 5 fraction was essentially that described by HOAGLAND, KELLER AND ZAMECNIK4. (b) The incorporation of 14C-labelled amino acids from Chlorella hydrolysate into the pH- 5 fraction was performed as described by FRASER AND HOLI)SWORTII5. The radioactivity associated with nucleic acid and protein that was precipitated by ice-cold 5 o, ,,o trichloroacetic acid was assumed to be a measure of amino acid activation.
Incorporation o~ amino acids into whole cells and protoplasts The conditions used were those described in the previous paper 1 except that when protoplasts were used the medium contained in addition IO °:o (w/'v) sucrose. When subcellulm fractions were prepared the incubation mixture was cooled in ice and the cells lysed as described.
Incorporation o/ amino acids into subcellular /ractions (a) The supernatant obtained at 2o ooo ×g (2.5 mg protein) was incubated with Io/,moles ATP, o.2 5/,mole GTP, 5/,moles phosphoenolpyruvate, 2opmoles MgC12, ISo/,moles KCI, 5o/*g casein hydrolysate, I/,C of 14C-labelled Chlorella hydrolysate (or [14Cjphenylalanine) in 3 ml o.i 3 M Tris-HC1 (pH 7.5)- The reaction was stopped by chilling in ice and addition of an equal volume of ice-cold IO % trichloroacetic acid. The materials were prepared for counting the radioactivity as previously describedL (b) Washed membranes, 15o-25o/~g protein, were incubated in I ml of the following medium: 2o/~moles KC1, 5o/,moles Tris, 5° pmoles maleic acid, 5 !,moles MnCl,,, I/1mole MgCI2, 2/,moles ATP, o.1/~mole of each GTP, UTP, CTP, dd\TP,.
Biochim. Biophys. Acta, ~23 (I906) 370-389
PROTEIN SYNTHESIS IN A THERMOPHILE
379
dGTP, dTTP and dCTP, 2 mg casein hydrolysate and I FC of Chlorella hydrolysate (or [14C]phenylalanine). After incubation for a given time at the experimental temperature the mixture was chilled in ice. From this point different procedures were employed. In some experiments the radioactivities incorporated into material stable to cold and hot trichloroacetic acid were measured 1. To determine the distribution of radioactivity within the membrane the incubation mixture was centrifuged at 20 o o o × g for 15 min and the membranes were resuspended in water and divided into two equal portions. The 20 o o o × g supernatant was treated with 5 °o trichloroacetic acid to yield any protein that had leached from the membranes. One portion of the membranes was mixed with an equal volume of 8o o/o (w,'v) phenol in water and the amino acid-tRNA complex prepared as described above. The second portion was treated successively with 9 vol. acetone, methanol (twice), chloroform and ether to give a lipid extract that was evaporated under a stream of N2. The lipid was dissolved in I ml ethanol and 5 ml petroleum ether and the organic layer washed three times with 2 ml water. The residue of protein left after lipid extraction was heated in a boiling-water bath with 2 ml I M NaCI. The saline extract was treated with 2 vol. ethanol and the precipitated RNA-amino acid complex collected. The residue from the saline extraction was treated with hot trichloroacetic acid and solvent as previously described I and the protein counted for radioactivity.
Heat stability o/membrane-activating enzymes A soluble extract was made from washed membranes by treatment in o.13 M Tris-HC1 (pH 7-5) containing I mM perfluorooctanoate with ultrasonic vibration for IO min (6o-W M.S.E. ultrasonic disintegrator) the membranes being immersed in ice during treatment. The material was then centrifuged at lO5 ooo ×g for 30 rain. The supernatant and the original membranes were compared for their heat lability by heating them for tile specified times at 60 °, then estimating the residual amino acid-activating activity by the incorporation of 14C-labelled amino acids into material precipitated by ice-cold trichloroacetic acid (as described above).
RESULTS
Studies with whole cells and protoplasts Whole cells were incubated with x4C-labelled Chlorella hydrolysate for 2 and IO min, the cells lysed as described and the distribution of radioactivity in the cell fragments determined. Table I shows that material sedimenting at 5000 ×g haci the greatest specific activity. As the time of incubation increased a significant amount of radioactivity appeared in the lO5 ooo ×g pellet and supernatant and some of this was stable to hot trichloroacetic acid, i.e. represents protein synthesis. The 5000 ×g pellet, which contained the most radioactivity, was examined further by distribution on a sucrose gradient. The pellet gave two main fractions. /o sucrose layer appeared to be cell One which collected in the upper part of the 30 o~ membranes and fragments of cell wall when examined in the electron microscope. Biochim. Biophys. Acta, x23 (1966) 376-389
380
B. BUBELA, E. S. HOLDSWORTH
TABLE I DISTRIBUTION OF IIC-LABELLED AMINO ACIDS IN CELL F R A C T I O N S
The i n c u b a t i o n m i x t u r e contained 2o ml of bacterial s u s p e n s i o n (A700m# = IO) in o.13 M T r i s HC1 (pH 7.5), 2o/zC of 14C-labelled a m i n o acids (Chlorella hydrolysate, ioo #C/mg), 2oo mg of glucose, 2o m g of casein h y d r o l y s a t e in a t o t a l 4 ° ml of o.13 M Tris-HC1 (pH 7.5). The material w a s i n c u b a t e d for io m i n at 63 °, cooled to 37 ° and lysed as described in t h e METHODS section. The p r o t o p l a s t s were disrupted b y osmotic shock and the resulting material w a s separated into individual fractions. The r a d i o a c t i v i t y was m e a s u r e d and expressed as #/~C/mg of m a t e r i a l insoluble in h o t a n d / o r cold trichloroacetic acid.
Fraction
2 rain
Whole cells 5ooo × g pellet 20 ooo × g pellet IOO ooo × g pellet IOO ooo × g s u p e r n a t a n t
IO rain
Cold trichloroacetic acid
Hot trichloroacetic acid
Cold trichloroacetic acid
Hot trichloroacetic acid
26o 445 65 8o 25
I9o 298 45 5° io
78o 133o 195 278 402
58o 93 ° 137 21o 28o
A smaller fraction which collected at the bottom of the 5o % sucrose layer had the highest specific activity and was rich in nucleic acid. The radioactivity associated with this heavy fragment was labile to treatment with hot trichloroacetic acid. Since the distribution of radioactivity in the lysed cell could be complicated by the presence of fragments of cell wall, experiments were made with protoplasts. After incorporation of amino acids and lysis of the protoplasts by osmotic shock, the subcellular fractions were prepared and the 5000 ×g pellet was further purified by distribution on a sucrose gradient as described for the preparation of
TABLE II MEMBRANE
PARTICIPATION
IN PROTEIN
SYNTHESIS
P r o t o p l a s t s p r e p a r e d as in the METHODS section were incubated in the presence of 1*C-labelled a m i n o acids at 63 °. The i n c u b a t i o n m i x t u r e contained I m l o f a suspension of p r o t o p l a s t s (A700m# = IO) in o.13 M Tris-HC1 (pH 7.5), i #C of 14C-labelled a m i n o acids (Chlorella hydrolysate, I o o # C / rag), IO m g of glucose, I m g o f casein h y d r o l y s a t e in t o t a l v o l u m e of 2 ml of IO % (w/v) sucrose in o.13 M Tris-HC1 (pH 7.5). One sample w a s i n c u b a t e d for 2o sec at 63 °, a second s a m p l e for 3 m i n and a t h i r d sample for 3 min, t h e n 3 mg of casein h y d r o l y s a t e were added and the reaction m i x t u r e i n c u b a t e d for a f u r t h e r 3 min. After the incubation, 5/*g of chloramphenicol were added to each sample, which w a s frozen immediately in a solid CO2-acetone freezing mixture. The p r o t o p l a s t s were disrupted b y adding the frozen material to 6 vol. of ice-cold water. The individual fractions were p r e p a r e d and the radioactivity in h o t a n d / o r cold trichloroacetic acid-insoluble material w a s measured. S y m b o l s N A and P represent nucleic acids and proteins, respectively.
Material
5000 × g pellet Membranes io ooo x g pellet 135 ooo × g pellet I35 ooo × g s u p e r n a t a n t
2o sec (minGling)
3 rain (mt~C /mg )
3 + 3 rain (ml~C /mg )
NA
P
NA
P
NA
P
o.612 o.712 O.lO 3 0.250 o.o96
0.298 0.385 o.o96 o. 112 o.121
1.o3o 1.65o o.2o 7 o.416 O.lO 7
o.5ol 0.652 o.125 o.2ol o.216
0.065 0.976 o.165 0.375 o.iio
0.572 o.765 o.137 o.135 1.o7o
Biochim. Biophys. Mcta, 123 (1966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
38I
membranes. From Table II it can be seen that the membrane fraction contained the greatest specific activity except where the radioactivity was "chased" into the lO5 ooo × g supernatant by further incubation with non-labelled amino acids. The material in the membrane fraction did in fact appear to be protoplast membranes as can be seen from the electron micrograph (Fig. i). In the 2o-sec incubation it appears that the membrane fraction was the first to become radioactive.
Incorporation o/ amino acids into subcellular /ractions Activation o/ amino acids. The formation of amino acid hydroxamates was taken as a measure of amino acid activation. Control tubes in which there were no added amino acids served to measure the small amount of hydroxamate formation from endogenous amino and fatty acids. Amino acid-activating systems are precipitated in the pH-5 fraction in mammalian systems ~ and it was interesting to find that the system in B. stearothermophilus could be precipitated in the same manner.
Fig. I. Electron micrographof protoplast membrane of B. stearothermophilus. method
Prepared by the
of ~ELLENBERG, RYTER AND SEEHAND 6.
Biochim. Biophys. Acta, 123 (1966) 376-389
382
B. BUBEI.A, E. S. HOI.I)SWORTtI
As an assurance t h a t a m i n o acid a c t i v a t i o n was being measured, e x p e r i m e n t s were m a d e to show t h a t the p H - 5 fraction could i n c o r p o r a t e ~4('-labelled a m i n o acids into m a t e r i a l p r e c i p i t a t e d b y ice-cold trichloroacetic acid. This m e t h o d includes a further s t e p after a c t i v a t i o n i.e. a t t a c h m e n t of the a m i n o acids to t R N A . The results of the two m e t h o d s are v e r y similar ;is shown in Fig. 2. A c t i v a t i o n was not r a p i d until 35 -4o° h a d been reached and rose to a m a x i m u m at 59 61°. I t was observed t h a t i n c u b a t i o n of the p H - 5 fraction above 45 ~ caused a p r e c i p i t a t e to form. "1"o s t u d y the t h e r m a l s t a b i l i t y of the amino a c i d - a c t i v a t i n g enzymes the pH-5 fraction was h e a t e d for IO min at various t e m p e r a t u r e s and then tested for its a c t i v a t i n g a c t i v i t y b y the h y d r o x a m a t e m e t h o d . F r o m the results shown in Fig. 3 it can be seen t h a t the enzymes are not stable a b o v e 4 o°. The p r e c i p i t a t e s t h a t formed were inactive when tested separately. The concentration of n u c M c acid in solution did not change during the incubation.
~
,°I
a"0 8
30
2o~
¢
0.4-~
@-
0
I0
20
30
40
50
60
Temperature Fig. 2. Effect on t e m p e r a t u r e on a m i n o acid a c t i v a t i o n by pH- 5 fraction. The a m o u n t of a m i n o acid h y d r o x a n , a t e ( O ) o r t h e a m o u n t of t4('-labelled a m i n o acid t R N A c o m p l e x ( O ) formed in xo rain a t d i f f e r e n t t e m p e r a t u r e s was d e t e r m i n e d as de s c ri be d in t h e .METtlOI)S section.
2.5
°°t o6t o
20
o4
rl
1.0
0.2
20
30 40 50 Temperature
60
0.5
Fig. 3. S t a b i l i t y of the a m i n o a c i d - a c t i v a t i n g e n z y m e s to he a t . pH - 5 e n z y m e w a s h e a t e d for I o m i n a t t h e specified t e m p e r a t u r e s and c e n t r i f u g e d . The a m i n o a c i d - a c t i v a t i n g activity, rem a i n i n g in s o l u t i o n was m e a s u r e d by the h y d r o x a m a t e m e t h o d ((3). P r o t e i n r e m a i n i n g in s o l u t i o n ( 0 ) was m e a s u r e d as d e s c r i b e d in tile METIIOIJS section.
Biochim. Biophys. A cta, r23 (t,466) 376-389
383
PROTEIN SYNTHIt'SIS IN A THERMOPHILE
Thermostability o/the amino acid-tRNA complex. The ~4C-labelled a m i n o a c i d t R N A complex was prepared a n d dissolved in o.13 M T r i s - H C l (pH 7-5) a n d heated at 37 ° a n d 63 ° for specified times. The r a d i o a c t i v i t y in material t h a t could be precipitated with ice-cold trichloroacetic acid was regarded as stable complex. Fig. 4 shows the results obtained. Approx. 5 ° % of the complex was decomposed in io min at 63 ° . Incorporation o/amino acids into 2o ooo × g supernatant. Lysed cells prepared b y using perfluorooctanoic acid were used to prepare a 2 o o o o × g s u p e r n a t a n t . I n c o r p o r a t i o n of ~4C-labelled a m i n o acid into this s u p e r n a t a n t , subfractions prepared from it and the reconstituted system are shown in Table 1lI. The results show t h a t incorporation at o ° is relatively high or alternatively, the increase in going from o ° to 63 ~ was only' two-fold. Chloramphenicol did not alter the a m o u n t of incorporation at o ° but completely inhil)ited the difference between o ° a n d ()3 °. Almost all of the limited ability to incorporate a m i n o acids was in the material t h a t precipitates at pH 5. Trans/cr o/amino acids/tom the tRNA complex into proteins. Since the fraction t h a t precipitates at pH 5 appeared to incorporate amino acids an a t t e m p t was made to see whether amino acid could be transferred from the 14C-labelled a m i n o a c i d t R N A complex (prepared as described in the .~fETHODS section) into hot trichloroacetic acid-stable material. Fig. 5 shows t h a t the transfer takes place rapidly at 63 °. Stvdy o/ ribosomes. A s t u d y of the ribosomes of B. stearothermophilus was made at two c o n c e n t r a t i o n s of Mg"'. The s e d i m e n t a t i o n values in o.I M NaCl plus o.oi M MgC12 were 31 S, 50 S, and 73 S, a n d in o.I M NaCI plus I mM MgC12 these values were 28 S, 48 S, a n d 65 S. The m a j o r portions were in the 73-S and 65-S fractions. No particles were seen t h a t could correspond to polysomes. 100. ~
~ t - ~ -
0
.
~ U
60-
~" 0.6'
,~ 40.
o ~0.4
c
0 Q.
~0.2 c
0
5 10 20 30 T~me (m~n)
40 Tirne (rain)
Fig. 4. Effect of heat on amino acid tRNA complex, liC-labelled amino acid-tRNA complex was prepared as described in the METHODSsection and heated at .37° (S) and (~3° (O) for the specified times. The radioactivity precipitable with cold trichloroacetic acid is a measure of unchanged complex and was expressed as per cent of the original complex. Fig. 5. Transfer of t4C-labelled amino acids from the tRNA complex to proteins, t4C-labelled amino acid-tRNA complex (prepared as in the METHODSsection) was incubated for io rain at 4°° (0) or 63 ° (O) in the presence of pH-5 fraction. 14C incorporated into material stable to hot trichloroacetic acid was regarded as protein.
Biochim. t3iophys. Acta, Iz3 (J966) 376-389
B. BUBELA, E. S. HOLDSWORTH
384 TABLE III INCORPORATION
OF AMINO
ACIDS
INTO
2 0 OOO X g S U P E R N A T A N T ,
ITS
SUBFRACTIONS
AND
A RE-
C O N S T I T U T E D S Y S T E M , A T O °, 3 7 ° A N D 6 3 °
The i n c u b a t i o n m i x t u r e contained 20 ooo × g s u p e r n a t a n t (2.5 mg of protein), io #moles of ATP, o.25 # m o l e of GTP, 5/*moles of p h o s p h o e n o l p y r u v a t e , 5 °/~g of casein hydrolysate, i/~C of llC-labelled anlino acids (Chlorella hydrolysate, i o o / , C / m g ) , 21/~moles of MgC12, i8o/~moles of KCI in 3 ml o.13 M Tris-HC1 (pH 7.5). W h e r e individual subfractions were investigated, separately or in a mixture, the a m o u n t p r e s e n t was similar to the original c o m p o s i t i o n of the 2o ooo × g s u p e r n a t a n t . The material was i n c u b a t e d for io rain at the e x p e r i m e n t a l t e m p e r a t u r e . The radioactivity was m e a s u r e d and expressed as /HzC/mg of material insoluble in h o t trichloroacetic acid.
Material
rng per fraction
o°
37 °
63 °
20 ooo X g s u p e r n a t a n t Ribosomes p H - 5 fractions pH- 5 supernatant R e c o n s t i t u t e d material
2.5 ° 0.05 2. io 0.35 2. 5
15.5 9.2 9.7 12.3 13.6
18. 7 9-5 12.5 12. 7 16.6
30.0 9.5 24.7 15.o 23.7
Role o/poly-U in protein synthesis in B. stearothermophilus. Since there seemed to be no polysomes as such in the 20 ooo × g supernatant, experiments were made to study the effect of poly-U on incorporation of phenylalanine. Table IV shows that poly-U stimulates the incorporation of phenylalanine into peptide bonds and the results also suggest that there is some destruction of poly-U during the IO rain at 63 ° since if the material was added in portions a greater incorporation was obtained. It was again noticed that during incubation a precipitate formed presumably due to denaturation of protein. Since the ionic strength of the buffer used in these experiments is high, o.13, the experiment was repeated in buffer with ionic strength o.o13 and the incorporation studied at various temperatures. Table V shows that optimum incorporation took place at 45 ° and again a precipitate formed at temperatures above 45 °. Cell-free extracts prepared by grinding protoplasts with alumina 7 gave very similar results. T A B L E IV INFLUENCE
OF POLY-UON
THE INCORPORATION OF PHENYLALANINE
I N T O 2 0 OOO N g S U P E R N A T A N T
The incorporating m i x t u r e contained 20 ooo × g of s u p e r n a t a n t (3 mg of protein), IO/~moles of ATP, 0.25/~mole of GTP, 5 # m o l e s of p h o s p h o e n o l p y r u v a t e , io/~g of casein hydrolysate, i #C of uniformly labelled phenylalanine (specific activity i o o # C / m g ) , i o # g of phenylalanine, 21 /~moles of MgCI~, 18o/~moles of KC1 in 3 ml o. 13 M Tris HC1 (pH 7.5). To one aliquot of the prepa r a t i o n i n c u b a t e d at 63 °, lOO/~g of poly-U were added. To a n o t h e r aliquot 25/~g of poly-U were added every 2.5 rain during t h e io min of incubation. The radioactivity of the material w a s m e a s u r e d and expressed a s / ~ # C / m g of material insoluble in h o t trichloroacetic acid,
Temperature
Radioactivity
Increase above o ° incorporation
o° 63 ° 63 ° + poly-U 6 3 ° + p o l y - U added in p a r t s
16. 7 35-3 42.5 50.2
-2. I-fold 2.5-fold 3.o-fold
Biochim. Biophys. Acta, 123 (1966) 376-389
PROTEIN SYNTHESIS IN A THERMOPHILE
385
TABLE V INFLUENCE OF POLY-~J ON THE INCORPORATION AT DIFFERENT TEMPERATURES FOR IO MIN
OF PHENYLALANINE
I N T O 2 0 OOO X g S I J P E R N A T A N T
M e t h o d as for T a b l e I V w i t h t h e e x c e p t i o n t h a t t h e s t r e n g t h of t h e T r i s - H C 1 buffer w a s O.Ol 3 M. Po ly-U, 25 pg, was a d d e d a t 2.5-min i n t e r v a l s .
Temperature
Radioactivity (IHzC/mg )
o° 1°0 20 ° 3 °0 35 ° 4 °0 45 ° 5° 55 ° 60 °
28. 3 29.7 31.9 37.9 56. 7 75.4 98.6 81.8 73.3 60.2
1.0
/
..EEO.S o ~EO.6
g o
~o
?
0.4
£0.2 ~,
~b203o
,~o
50
40
Ternperoture
Fig. 6. I n c o r p o r a t i o n of a m i n o acids i n t o m e m b r a n e s . M e m b r a n e s p r e p a r e d from p r o t o p l a s t s of B. stearothermophilus were i n c u b a t e d w i t h 14C-labelled C hl ore l l a h y d r o l y s a t e as d e s c r i b e d in t h e METHODS section. The r a d i o a c t i v i t y i n c o r p o r a t e d i n t o m a t e r i a l s t a b l e t o cold ( O ) or h o t ( 0 ) t r i c h l o r o a e e t i c acid w a s m e a s u r e d a n d e x p r e s s e d as m/zC/mg of nucleic a c i d or p r o t e i n , r e s p e c t i v e l y .
1.0-
~
0.8 g .~0.6 ~0.4
o.2
10
20 30 40 Temperature
50
60
Fig. 7, I n c o r p o r a t i o n of a m i n o a c i d s i n t o m e m b r a n e s i n t h e p r e s e n c e of m R N A . E x p e r i m e n t a l : o n d i t i o n s as for Fig. 6 w i t h t h e i n c l u s i o n of m R N A i n t h e i n c u b a t i o n m i x t u r e . T h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o m a t e r i a l s t a b l e to cold ( O ) or h o t ( O ) t r i c h l o r o a c e t i c a c i d w a s m e a s u r e d a n d ~xpressed as mffC/mg of nucleic a c i d or p r o t e i n , r e s p e c t i v e l y .
Biochim. Biophys. Acta, 123 (1966) 376-389
386
B.
BUBELA,
E.
S. H O L D S W O R T H
Membranes as a site o~ protein synthesis It was reported b y S P I E G E L M A N 9 that isolated membranes from Escherichia coli incorporate amino acids into hot trichloroacetic acid-insoluble material. When membranes from B. stearothermophilus were incubated as described by SPIEGELMAN9 the results obtained in Fig. 6 were obtained. The 14C-labelled amino acids became attached mainly to nucleic acids, i.e. material unstable to hot trichloroacetic acid and there was only a small incorporation into protein. If the incubation mixture was supplemented with RNA extracted from B. stearothermophilus the situation was changed (Fig. 7) and amino acids became incorporated into protein. The optimum temperature for these incorporations was 65 ° , although the membranes tended to flocculate at temperatures above 45 ° . The incorporation of amino acid into membranes was studied in more detail b y following the distribution of radioactivity in lipids, attached to nucleic acid, or incorporated into protein, as affected by time of incubation. It is apparent that the highest specific activity appears firstly in the lipid fraction (Table VI), then in the amino acid-tRNA complex and finally in the membrane protein. Some radioactivity protein also appeared in free solution in the incubation mixture. Repeated extraction of the lipo-amino-acid complex in petroleum ether solution with I o/~} (w/v) aqueous solution of casein hydrolysate did not remove the radioactivity into the aqueous phase. The lipo-amino-acids were examined by chromatography on W h a t m a n 3 MM paper using benzene-n-butanol (I : I, v/v) saturated with water as a solvent. The lipo-amino-acids had R F ' s between 0. 4 and 0. 7 in this solvent whereas the free amino acids had RF's below o.15. TABLE
VI
INCORPORATION
OF AMINO
ACIDS
INTO
MtgMBRANES
AT 6 0 ~:
M e m b r a n e s w e r e i n c u b a t e d f o r 3 ° sec, I r a i n a n d 3 r a i n i n t h e m e d i u m d e s c r i b e d f o r F i g . 7. T h e v a r i o u s f r a c t i o n s w e r e p r e p a r e d a s d e s c r i b e d i n t h e METHODS s e c t i o n . T h e r a d i o a c t i v i t y is e x p r e s s e d a s m # C / m g o f R N A .
Fraction RNA-amino acid (phenol method) membrane RNA amino acid Lipo amino acid Membrane protein Supernatant protein
3 o sec
i rain
3 rain
7.8
lO. 5
13.2
-15. 3 i.i --
-i8.i 2.6 --
15. 3 "23.3 lO.2 7- i
Thermostability o/ the activating enzymes o~ membranes The incorporation of amino acids into protein by the membranes was found to decrease only 20 % after heating for IO rain at 63 ° (ref. IO). A comparison was made of the heat stability of the amino acid-activating system in membranes with a soluble activating system prepared from the membranes. The results shown in Fig. 8 indicate that the soluble activating system prepared from the membranes was less stable to heat than that present in the original membranes. No significance can be attached to the difference in specific activity between the two preparations since they had different proportions of protein to RNA. Biochim. B i o p h y s . Acta, 123 ( 1 9 6 6 ) 3 7 6 - 3 8 9
PROTEIN SYNTHESIS IN A THERMOPHILE
387
looJ 90-
8o! 7e4 %60 50 40 3O
oi 2
4 6 Time (rain)
8
10
Fig. 8. H e a t s t a b i l i t y of a m i n o a c i d - a c t i v a t i n g s y s t e m s . M e m b r a n e s p r e p a r e d f r o m p r o t o p l a s t s of B. stearothermophilus ( 0 ) a n d a soluble p r e p a r a t i o n p r e p a r e d b y u l t r a s o n i c t r e a t m e n t of t h e m e m b r a n e s ( 0 ) were h e a t e d a t 63 ° for t h e specified times. T h e residual a m i n o a c i d - a c t i v a t i n g a c t i v i t y ( m e a s u r e d b y i n c o r p o r a t i o n of laC-labelled a m i n o acid into m a t e r i a l p r e c i p i t a t e d b y cold trichloroacetic acid, see t h e METHODS Section) w a s e x p r e s s e d as a p e r c e n t a g e of t h e original activity.
DISCUSSION
Whole cells of B. stearothermophilus have been shown to incorporate 14Clabelled amino acids into protein with an optimum temperature of 60 ° (ref. I). When cells prepared in this way were disintegrated and separated into fractions b y centrifugation, it was found that the highest specific activity was located in membranes (Table I and Fig. I). Protein synthesised b y the membranes m a y become part of the soluble cytoplasm since the radioactivity incorporated into membranes could be ,,chased" into the soluble part by further incubation with non-labelled amino acids (Table II). The supernatant obtained at 20 ooo ×g from lysed cells of B. stearothermophilus contained an amino acid-activating system that had an optimum activity of approx. 60 ° using an incubation of IO rain. It was curious to find that activation of amino acids was very limited until temperatures above 35 ° were used. In these experiments there is no question of transport of amino acid to the site of activation since the activating system was soluble. Calculation of the heat of activation from the data in Fig. 2 gave a figure of 12 ooo cal, which is similar to that calculated for the incorporation of amino acids ] . Therefore, this is another fragment of evidence that the activation of enzymes from this organism requires higher temperatures than enzymes from mesophilic bacteria. This aspect was discussed in the previous paper ]. A_bove 4 °° the activating system was heat labile and 5o %was destroyed b y h e a t i n g ro min at 6o °. That the amino acids were activated was shown by the isolation of an ~mino a c i d - t R N A complex. This complex heated in buffer (with absence of enzymes) was thermolabile. A similar observation was made on the isolated leucine-tRNA =omplex from B. stearothermophilus by ARCA et al. ]1. An unexpected finding was that pH-5 fraction prepared from a lO5 ooo ×g ;upernatant of lysed cells, therefore presumably free from ribosomes, could transfer 4C-labelled amino acids from their complex with t R N A to material stable to hot :richloroacetic acid. The pH-5 fraction in fact constituted the most active part of the ,'o ooo × g supernatant when this was investigated for its ability to incorporate amino tcids into protein. However, the ability of the 2o ooo ×g to incorporate amino acids Biochim. Biophys. Acta, 123 (1966) 376-389
388
B.t'~UBF.I.A, IL. S. HOI.DS',A'ORTH
into protein was v e r y low c o m p a r e d with t h a t of whole ce.lls or protoplasts. One reason for this m a y be t h a t we could find no polysomes in our p r e p a r a t i o n . The largest particles were ribosomes of 7o S. MANGIANTINI et al) 2 r e c e n t l y r e p o r t e d t h a t particles of ~oo-S t y p e only a p p e a r e d in e x t r a c t s from B. stearothermotbhilus in Tris buffer c o n t a i n i n g 5 mM m a g n e s i u m acetate. The work on t u r n o v e r of nucleic acids r e p o r t e d in the previous p a p e r ~ showed t h a t labile nucleic acid had a life of only I min. Therefore, the absence of polysomes was not unexpectecl. Even when the 2o ooo x g s u p e r n a t a n t was s u p p l e m e n t e d with poly-U the incorporation, although s t i m u l a t e d , was still v e r y small. FRIEI).~IAN AND \VI.;INSTEIX la also found poly-Us t i m u l a t e d p h e n y l a l a n i n e i n c o r p o r a t i o n in a cell-free e x t r a c t of /4. stearothermophilus. T h e o p t i m u m t e m p e r a t u r e for i n c o r p o r a t i o n of p h e n y l a l a n i n e in this system was 45 ° a n d in m a n y e x p e r i m e n t s it was noticed t h a t heating the 2o ooo × g s u p e r n a t a n t a b o v e 45 ° gave a p r e c i p i t a t e of protein. Thus it a p p e a r s t h a t the isolated cell c o n t e n t s arc'. u n s t a b l e to heat. Ribosomes from B. stearolhermophilus have recently been shown to be more stable to heat t h a n those from E. coli L2, a l t h o u g h the tests were of physical p r o p e r t i e s and not of their function in protein synthesis. The most i m p o r t a n t site of a m i n o acid i n c o r p o r a t i o n a p p e a r s to be in the m e m b r a n e s o b t a i n e d from p r o t o p l a s t s of /4. stearothermophilus. This agrees with the obs e r v a t i o n s m a d e with whole cells. Cell m e m b r a n e s t h a t h a d been washed three times with o.I3 1~1 Tris HCI (pH 7.5) a n d were p r e s u m a b l y free from soluble proteins, were able to a c t i v a t e a m i n o acids a n d transfer t h e m to m a t e r i a l stable to t r e a t m e n t with hot trichloroacetie acid, i.e. proteins. The m a j o r incorporation was into an amino acid--. R N A c o m p l e x w h i c h was e x t r a c t e d and shown to contain r a d i o a c t i v i t y . W h e n crude. m R N A was included in the i n c u b a t i o n m i x t u r e for the m e m b r a n e s , much more a m i n o acid was i n c o r p o r a t e d into protein. The. o p t i m u m t e m p e r a t u r e for incorporation into m e m b r a n e s was ~5 ~. In c o n t r a s t to the 2o o o o × g s u p e r n a t a n t there was a large i n c r e m e n t of i n c o r p o r a t i o n between o ~' and 6o °, also the a m o u n t of i n c o r p o r a t i o n o b t a i n e d when using m e m b r a n e s was comparalfie with t h a t o b t a i n e d with p r o t o p l a s t s . M e m b r a n e s could be h e a t e d for 2o min at 65 ° with the loss of only 2o
Biochi~z. IJiophys. Acta, 1, 3 (tc~o0)37~J-~S9
PROTEIN SYNTttESIS IN A THERMOPHILE
389
thesis, stated that there was no proof that they were obligatory intermediates in protein synthesis. The complexes may be transport forms of amino acids or a storage form of the activated amino acids. There ix a growing volume of evidence that the cytoplasmic membrane ix an important site of protein synthesis in bacteria e.t,. in 13. megateri.um 14, in t:.'. colig, le, in B. subtilis :~, in Streptococcus /aecalis 1~. That this is indeed protein synthesis is suggested by the observation 10 that a membrane preparation from E. coli would produce alkaline phosphatase. In B. stearothermophilus not only is the membrane the most active site for the incorporation of amino acids but the results indicate that the mechanism for protein synthesis is more stable to heat when it ix part of the membrane. Several observations in this and the accompanying paper 1 may have a bearing on the ability of 13. stearothermophilus to grow at high temperatures. Firstly the energy of activation of the enzymes in thermophiles may be higher than in mesophiles, although not enough enzymes have been studied. Secondly, the turnover of protein and nucleic acid 1 is more rapid in B. stearothermophilus than in E. coli thus more rapid repair of heat damage ix possible. Thirdly, it seems that organisati()n into membrane-type structures can confer a certain amount of heat stability on enzymes. Further observations on this aspect will be presented in a subsequent paper.
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Biochim. Biophys. Acta, i23 (1966) 376-389