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Purification and properties of the prolyl RNA synthetase of Escherichia coli
ARCHIVES
OF
BIOCHEMISTRY
Purification
AND
and
106, 147-152 (1964)
BIOPHYSICS
Properties
of the
Prolyl
of Escherichia
RNA
Synthetase
cob’
S. J. NORTON From the Department
of Chemistry, Received
North
Texas State University,
November
Denton,
Texas
29, 1963
A prolyl RNA synthetase isolated from sonic extracts of Escherichia coli 9723 has been purified approximately 20.fold using conventional enzyme purification techniques. The purified enzyme catalyzes both an L-proline-dependent adenosine triphosphate-inorganic pyrophosphate exchange and a transfer of L-proline-Cl4 to soluble ribonucleic acid (sRNA). DL-3,4-Dehydroproline competitively inhibits the transfer of prolineXY to sRNA, but does not inhibit the proline-dependent ATP-PPi exchange. Rather, a 3,4-dehydroproline-dependent ATP-PPi exchange catalyzed by the prolyl RNA synthetase can be demonstrated. A 4-hydroxy-L-proline-dependent ATP-PPi exchange is also catalyzed by the enzyme when high concentrations of this imino acid are employed. Hydroxyproline at relatively high concentrations is not an effective inhibitor of the transfer of proline-Cl4 to sRNA. Some of the properties and conditions for optimal activity of the purified enzyme are reported. INTRODUCTION
A great volume of evidence has accumulated that in the process of protein biosynthesis amino acyl RNA synthetases play an intermediary role by activating and transferring amino acids to specific soluble ribonucleic acid molecules. These enzymes have been shown to catalyze an amino acid-dependent adenosine triphosphate-inorganic pyrophosphate exchange, suggesting the probable formation of amino acyl adenylateenzyme complexes. Further, in experiments employing U4-labeled amino acids, these enzymes have been shown to catalyze the formation of amino acyl RNA complexes (1). Several amino acyl RNA synthetases have been purified and studied (2-5); however, other than evidence for its existence (6), no detailed investigation has been made on a prolyl RXA synthetase.2 1 This work was supported in part by a research grant from the Robert A. Welch Foundation of Texas. 2 Subsequent to the preparation of this manuscript, a paper was published relating to the partial purification of a prolyl RNA synthetase from
In the course of an investigation of some aspects of proline metabolism in certain microorganisms, it was found desirable to study the nature of the reaction catalyzing the activation of proline for its subsequent incorporation into protein. It was therefore undertaken to purify and study the properties of the prolyl RNA synthetase from Escherichia coli, and to test the effect(s) of 3,4-dehydroproline (7)) a known proline antagonist in certain microorganisms, on the activity of the purified enzyme. Herein are reported the results of this investigation. MATERIALS
AND
METHODS
MATERIALS Soluble ribonucleic acid from E. coli was obtained from General Biochemicals, Chagrin Falls, Ohio. uL-ProlinecarboxyllC’4 was obtained from California Corporation for Biochemical Research. L-Proline-U-Cl4 was obtained from Nuclear Chicago Corporation. Calcium phosphate gel was prepared by a previously described procedure (8). rat liver. The enzyme from rat liver differs in several aspects from the enzyme reported herein (64.
147
148
NORTON
The DEAE-cellulose, Type 20, was obtained from Brown and Company. Potassium P3aP? was prepared by pyrolysis of potassium P? for 1 hour, and appropriately diluted samples were used without further purification. DL-3,4-Dehydroproline was kindly supplied by Drs. Robertson and Witkop, National Institutes of Health. The author is deeply greatful to Mr. Morris Key andMrs. Sandra Scholes for their capable technical assistance during the course of this work. ENZYME
ASSAYS
For the measurement of proline-dependent ATP-PPi exchange the complete reaction mixtures contained in 1 ml: L-proline, 5rmoles; Tris buffer, pH 7.5, 100 Fmoles; potassium fluoride, 50 pmoles; sodium pyrophosphate-Pa*, 1 rmole (lOO,OOO800,060 cpm); and an appropriate amount of the enzyme fraction. Blanks containing no proline were included in each assay, and the exchange activity due to the presence of proline was obtained by difference. The reaction mixtures were incubated for 10 minutes at 37”C, and the reactions were terminated by the addition of 0.5 ml of 7% perchloric acid. After the ATP had been adsorbed and eluted from acid-washed charcoal by the method of Berg (2), aliquots were plated on concentric ring planchets and dried. Radioactivity was measured by use of a Nuclear Measurements Corporation Proportional Counter. After correction for background radioactivity the percentage and rate of exchange were calculated by the method of Davie et al. (9). The specific activity of the enzyme is reported as the number of micromoles of pyrophosphate exchanged per milligram of protein per hour. For the assay of the formation of prolyl RNA the complete reaction mixtures contained in 0.5 ml: L-proline-U-CY4, 6 mrmoles (320,009 cpm), or nn-proline-carboxyl-Cr4, 35 mpmoles (220,000 cpm); sRNA (excess), 1.5 mg; potassium ATP, 0.4 pmoles; magnesium chloride, 10 Fmoles; potassium phosphate buffer, pH 6.8, 50 rmoles; and a rate-limiting amount of the enzyme fraction. The reaction mixtures were incubated for 10 minutes at 37”C, and the reactions were terminated by the addition of 3 ml of cold 67% ethanol containing 0.5 M NaCl. Washing of the sRNA precipitate was fashioned after the procedure of Berg et al. (10). The washed sRNA was then dissolved in 1 ml of water, a 0.5 ml aliquot was plated and dried, and the radioactivity was determined. When either sRNA or enzyme was omitted from the reaction mixtures, the radioactive levels were only slightly above that of background. The specific activity of the enzyme is reported as the number of millimicromoles of L-prolyl-RNA formed per milligram of protein per hour.
Protein concentrations of the cell-free extracts were determined spectrophotometrically by the method of Warburg and Christian (11). Crude cell-free extracts (sonic extracts and ammonium sulfate fractions) were always dialyzed prior to assay against 0.02M potassium phosphate, pH 6.8, containing 0.002M magnesium chloride and 0.01 iVl p-mercaptoethanol. PREPARATION
OF CELL
EXTRACTS
Cells of E. coli 9723, grown in 5 ml of saltsglucose medium (12) for 14 hours at 37”C, were used to inoculate 100 ml. of a sterile medium which contained; KHzP04 , 17.0 gm; KzHPOa , 21.8 gm; yeast extract, 10 gm; and glucose, 10 gm per liter. This culture, after incubation for 8 hours at 37”C, was used t.o inoculate 6 liters of the above enriched medium. The culture was then incubated for 18 to 20 hours at 37°C and was harvested with a Sharples centrifuge. Usually 20 to 25 gm wet weight of cells were obtained under the above conditions. The cells were then washed and resuspended in 65 to 70 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.002 M magnesium chloride and 0.01 M fi-mercaptoethanol. This suspension in 25 ml. portions was then exposed to sonic oscillation for 3 minutes using a Branson Sonifier. Undisrupted cells and cell debris were removed by centrifugation at 30,000 g for 20 minutes at 4°C. The sonic extract was stored at 4”C, and all subsequent operations were conducted at 0” to 4°C. PURIFICATION
OF PROLYL
RNA SYNTHETASE
To 58 ml. of sonic extract containing 18.2 mg protein per milliter was added 5.8 ml of a 10% solution of streptomycin sulfate. After stirring 15 minutes, insoluble material was removed by centrifugation. To the supernatant solution (58 ml) a saturated solution of ammonium sulfate was slowly added to give a solution 28% saturated with respect to ammonium sulfate. After stirring for 15 minutes the precipitated protein was removed by centrifugation at 20,OOOg. The residue was discarded and the supernatant solution was then made 45y0 saturated by addition of saturated ammonium sulfate solution. After stirring for 15 minutes the insoluble protein was removed by centrifugation as previously and dissolved in 15 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.902 M magnesium chloride and 0.01 M p-mercaptoethanol. This ammonium sulfate fraction was then dialyzed overnight in 2 liters of the same buffer to give 19.5 ml of solution containing 14.1 mg protein per milliliter. The dialyzed 28 to 45oj, ammonium sulfate fraction (18 ml) was then treated with 88 mg of calcium phosphate gel (0.35 mg of gel per milligram
PROLYL
RNA
SYNTHETASE
149
rate of a proline-dependent ATP-PPi exchange. In early studies using crude cellfree extracts, attempts were made to demonstrate a proline-dependent hydroxamate formation in the presence of hydroxylamine; however, no such formation could be shown. This was not surprising in view of findings that measurement of amino aciddependent hydroxamate formation is too insensitive for the assaying of most amino acyl RiSA synthetases (13). Due to high pyrophosphatase activity in the crude enzyme extracts, it was necessary to add potassium fluoride (which effectively inhibited this activity) to the reaction mixtures when the pyrophosphate exchange assay was employed. Virtually all the pyrophosphatase activity was separated from prolyl RNA synthetase activity by selective adsorption on and elution from calcium phosphate gel (Table I, step III). The crude extracts were always dialyzed overnight before assaying. Even after extensive dialysis the crude extracts catalyzed an appreciable prolineindependent ATP-PPi exchange. However, the stimulation of the exchange activity upon addition of proline to the reaction mixtures was both significant and consistent. As the proline-dependent ATP-PP i exchange activity was purified the proline-independent ATP-PPi exchange activity was greatly decreased. As summarized in Table I, a purification of the prolyl RNA synthetase activity of approximately 20-fold has been obtained enzyme purification using conventional techniques. Enzyme fractions at each stage of purification were assayed by both prolinedependent ATP-PP i exchange and transfer of proline-Cl4 to sRNA. Both assay techniques gave good agreement with respect to the relative increase of enzyme specific activities at each purification step. In order that there be no doubt that the enzyme under investigation is actually a proline-activating enzyme, both DL-prolinecarboxyl-Cl4 and L-proline-U-Cl4 (obtained RESULTS AND DISCUSSION from different sources) were tested in assays For the assay for the presence of a prolyl involving transfer to sRT\‘A. Calculations of RNA synthetase in extracts of E. coli 9723, enzyme-specific activities were the same two procedures were employed; namely, the regardless of which proline substrate was measurement of transfer of CY4-labeled pro- employed. Further evidence was obtained line to sRNA and the measurement of the by use of paper chromatography. Enzyof protein). The pH was adjusted to 6.0 with 1 N acetic acid, the slurry was stirred for 15 minutes, and the gel was separated by centrifugation. Most of the desired enzyme activity was adsorbed on the gel by this procedure. The gel was washed by stirring with 20 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.002 M magnesium chloride and 0.01 M p-mercaptoethanol, and removed by centrifugation. The desired enzyme activity was eluted from the gel by stirring 15 minutes with 20 ml of 0.03 M potassium phosphate, pH 7.5, containing 0.002 M magnesium chloride and 0.01 M fl-mercaptoethanol. The gel was removed by centrifugation, and the supernatant solution (20 ml containing 1.8 mg protein per milliliter) was recovered. Most of 6he desired enzyme activity was eluted at this stage. Further elution with more concentrated buffer solutions failed to yield additional enzyme activity. Further purification of the prolyl RNA synthetase was obtained by use of a DEAE-cellulose column. The calcium phosphate gel eluate (15 ml) was passed through a column 1 cm in diameter containing 250 mg of DEAE-cellulose that had been previously washed with dilute hydrochloric acid and dilute sodium hydroxide and equilibrated with 0.03 M potassium phosphate, pH 7.5, containing 0.01 M fl-mercaptoethanol and 0.002 M magnesium chloride. After the gel eluate had passed through, the column was washed with 10 ml. of the same buffer. The prolyl RNA synthetase activity was recovered by successive additions to the column of 0.05, 0.07, 0.08, 0.09, 0.12, and 0.15 M potassium phosphate buffers, pH 7.5, containing the usual concentrations of P-mercaptoethanol and magnesium chloride. Fractions of 5 to 8 ml were collected and assayed. The desired enzyme activity was eluted from the column at buffer concentrations of 0.07 to 0.09 M. Active fractions were combined to yield 20 ml of solution containing 0.2 mg of protein per milliliter. The purified enzyme loses only a small amount of catalytic activity on storage at 4” C for one month, while complete loss of activity results upon exposure to a temperature of 60” C for a 5 minute period. Reducing conditions are necessary to prevent loss of prolyl RNA synthetase activity; therefore, the enzyme solutions were maintained in the presence of fi-mercaptoethanol throughout the purification procedure.
150
NORTON
PURIFICATION
OF
TABLE
I
PROLYL
RNA
SYNTHETASE Specific
Enzyme
fraction
I. Sonic extract II. Ammonium sulfate fraction 28-45% III. Calcium phosphate gel eluate IV. DEAE-cellulose
Yield (%I
100 60
Transfer to sRNAa (mpmoles/ Ww protein)
1.5 3.8
Activity ATP-PPi exchange* (pm&s/ hr/w protein)
0.08 0.20
25
10
0.40
10
30
1.8
a The reaction mixture contained: nn-proline, 35 mmoles, 2.2 X lo6 cpm; sRNA, 1.5 mg; potassium ATP, 0.4 pmole; magnesium chloride, 10 rmoles; potassium phosphate, pH 6.8, 50 pmoles; and a rate-limiting amount of each enzyme fraction. The reaction mixture volume was 0.5 ml and incubation was for 10 minutes at 37”. * The reaction mixture contained: L-proline, 5 pmoles; potassium ATP, 3 pmoles; magnesium chloride, 10rmoles; Tris buffer, pH 7.5,lOOrmoles; potassium fluoride, 50 pmoles; sodium pyrophosphate-Paz, 1 pmole, 8 X lo5 cpm; and a rate-limiting amount of each enzyme fraction. The reaction mixture volume was 1.0 ml and incubation was for 10 minutes at 37”.
matically produced Cr4-labeled sRNA was subjected to mild alkaline hydrolysis at 37”C, and the sRNA was then precipitated with ethanol. After concentration, the superC14-labeled natant solution (containing amino acid) was chromatographed on paper along with unlabeled n-proline in several solvents. In each instance the Rf value of the radioactive spot corresponded to that of the unlabeled proline. Some of the conditions for optimal activity of the prolyl RNA synthetase were studied. Variation of the pH between 6.8 and 7.6 had little effect on enzyme activity with either the exchange or the transfer reactions. Tris buffer, pH 7.5, was used with the pyrophosphate exchange assay, since phosphate buffer caused some precipitation in the reaction mixtures. Potassium phosphate buffer, pH 6.8, was employed with the transfer assay since there was the possibility of some hydrolysis of the enzy-
matically produced prolyl RNA at higher pH values. Reaction rates were linear with time for 10 minutes with both assay procedures. The effect of increasing concentrations of ATP on the formation of CY4-prolyl RNA was determined. From a LineweaverBurk plot of the data obtained (Fig. 1) the apparent K, value for ATP was found to be 6 X 1O-4 M. Concentrations of ATP greater than 0.6 pmoles per milliliter inhibited the transfer. It has previously been reported (7) that DL-3 ,4-dehydroproline inhibits the growth of several microorganisms including E. coli 9723, and that the toxicity is reversed in a competitive manner by n-proline. It was therefore of interest to test the effects of this inhibitor on the activity of the partially purified prolyl R3A synthetase, since the inhibition of proline activation would certainly result in cessation of cellular growth. As shown in Fig. 2, DL-3 ,4-dehydroproline
0
I
2
3
4
:
I/lSl x 10-S VATP, moles per liter FIG. 1. Lineweaver-Burk plot of the effect of ATP concentration on the transfer of n-proline-Cl” to sRNA. Reaction velocity (v) is in terms of moles per liter of prolyl RNA formed per second. The reaction mixture contained in a volume of 0.5 ml: L-proline-U-C14, 6 mpmoles, 3.2 X lo5 cpm; sRNA, 1.5 mg; magnesium chloride, 10 Imoles; potassium phosphate, pH 6.8, 50 rmoles; enzyme (DEAEcellulose fraction), 66 rg; and potassium ATP, 0.1 to 0.6 rmoles. Incubation was 10 minutes at 37°C.
PROLYL
RNA
inhibits prolyl RNA synthetase activity in a competitive manner. The apparent K, value for L-proline was calculated and found to be 1.2 X 1O-4 M. The apparent Ki value for 3,4-dehydroproline (based on the amount of ~-3 ,Pdehydroproline present) was calculated to be 12 X 10h4M. Experiments similar to that shown in Fig. 2 were run’ several times, and in each case the inhibition exhibited by the analog was competitive with proline. Thus in the intact microorganism, it is quite probable that at least one of the sites of inhibition of proline metabolism by DL-3,4-dehydroproline is at the site of proline activation. The transfer of amino acids to sRn’A molecules is a two-step process, the first being the formation of an amino acyl ade-
151
SYNTHETASE TABLE
16-
AND
EXRNA
Racii;.ci;ity Ra$mc$tity
None L-Praline 5pmoles L-Pro,ine’ 5 rmo,es + n;-3,4-dehydroproline, fimoles nL-3,4-Dehydroproline, 5 rmoles
(cpm)
(corrected for no praline control) (cpm)
875 3020 2905
0 2145 2030
1575
700
2940
2065
2820
1945
1190
315
1520
645
15
DL-3,4-Dehydroproline, 15 rmoles L-Proline, 5 rmoles 4-hydroxy-Lf proline, 20 Mmoles 4-Hydroxy-L-proline, 10 .umoles 4-Hydroxy-L-proline, 20 pmoles
18-
II
THE EFFECTS OF DL-3,kDEHYDROPROLINE 4-HYDROXY-L-PROLINE ON THE ATP-PPi CHANGE CATALYZED BY PURIFIED PROLYL SYNTHETASE
a The reaction mixture contained in 1 ml.: potassium ATP, 3 pmoles; magnesium chloride, 10 rmoles; Tris buffer, pH 7.5, 100 pmoles ; sodium pyrophosphate-P32 4.6 X lo5 cpm, 1 pmole; and enzyme (DEAE-cellulose fraction), 30 ,q. Incubation was for 10 minutes at 37°C.
Km for Proline
=
1234567 I/K1 x to-4 I/ Proline,
moles per liter
FIG. 2. Lineweaver-Burk plot showing the competitive inhibition of prolyl RNA synthetase activity by DL-3,4-dehydroproline. Conditions were as described under Fig. 1 except that 0.4 Fmoles of potassium ATP and 15 fig of enzyme (DEAE-cellulose fraction) were employed. The inhibitor concentration was 0.35 rmoles per 0.5 ml. O-O, L-proline; O-O, L-proline $ DL-3,4dehydroproline.
nylate-enzyme complex, the second being the transfer of the activated amino acid to a specific sRNA molecule. Inhibition of the over-all process can occur at either of these intermediate reaction processes (1). It was of interest, therefore, to determine whether the prolyi RNA sykthetase is capable of catalyzing a 3,4-dehydroproline-dependent ATP-PPi exchange or whether the exchange reaction is inhibited by the presence of this analog. As shown in TabIe II, DL-3,4-dehydroproline has no significant inhibitory action on the proline-dependent ATP-PPi exchange, while the analog does itself promote an ATP-PPi exchange. It has been reported (14) that in E. coli exogenously supplied 3,4-dehydroproline is incorporated into abnormal protein molecules in place of proline. Results obtained in this study are
NORTON
compatible with this finding. It is possible that 3,4-dehydroproline is transferred to the appropriate sRNA molecule as is L-proline, and the inhibition of transfer of L-proline-Cl4 to sRNA exerted by the analog (Fig. 2) is the result of its competition with L-proline for activation by the enzyme. As seen in Table II, 4-hydroxy-L-proline at high concentration also promotes an ATP-PPi exchange catalyzed by the prolyl RNA synthetase. Hydroxyproline in the presence of proline has no significant effect on the exchange reaction. In other experiments, hydroxyproline, at concentrations as high as 4 Itmoles per milliliter of reaction mixture, was found to have no significant effect on the transfer of proline-Cl4 to sRNA. It is probable that very high concentrations of hydroxyproline would be required to inhibit the normal catalytic function of the prolyl RNA synthetase of E. coli 9723. REFERENCES 1. BERG, P., Ann. Rev. Biochem. 30, 293 (1961). 2. BERG, P., J. Biol. Chem. 222, 1025 (1956). 3. SCHWEET, R. S., AND ALLEN, E. H., J. Biol. Chem. 233, 1104 (1958).
4. BERGMANN, F. H., BERG, P., AND DIECKMANN, M., J. Biol. Chem. 236,1735 (1961). 5. NORTON, S. J., RAVEL, J. M., LEE, C., AND SHIVE, W., J. Biol. Chem. 236, 269 (1963). 6. PETERKOFSKY, B., AND UDENFRIEND, S., Federation Proc. 21, 169 (1962). 6a. FRASER, M. J., AND KLASS, D. B., Can. J. Biochem. Physiol. 41, 2123 (1963). 7. SMITH, L. C., RAVEL, J. M., SKINNER, C. G., AND SHIVE, W., drch. Biochem. Biophys. 99, 60 (1962). 8. WOOD, W. A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 214. Academic Press, New York, 1955. 9. DAVIE, E. W., KONIGSBERGER, V. V., AND LIPMANN, F., Arch. Biochem. Biophys. 66,21 (1956). 10. BERG, P., BERGMANN, F. H., OFENGAND, E. J., AND DIECKMANN, M., J. Biol. Chem. 236, 1726 (1961). 11. WARBURG, 0.: AND CHRISTIAN, W., Biochem. 2. 310, 384 (1941). 12. ANDERSON, E. H., Proc. Natl. Acacl. Sci. U. S. 32, 120 (1946). 13. LOFTFIELD, R. B., AND EIGNER, E. A., Biochim. Biophys. Acta 72, 372 (1963). 14. FOWDEN, L., NESLE, S., AND TRISTR.~M, H., Nature 199, 35 (1963).
OF
BIOCHEMISTRY
Purification
AND
and
106, 147-152 (1964)
BIOPHYSICS
Properties
of the
Prolyl
of Escherichia
RNA
Synthetase
cob’
S. J. NORTON From the Department
of Chemistry, Received
North
Texas State University,
November
Denton,
Texas
29, 1963
A prolyl RNA synthetase isolated from sonic extracts of Escherichia coli 9723 has been purified approximately 20.fold using conventional enzyme purification techniques. The purified enzyme catalyzes both an L-proline-dependent adenosine triphosphate-inorganic pyrophosphate exchange and a transfer of L-proline-Cl4 to soluble ribonucleic acid (sRNA). DL-3,4-Dehydroproline competitively inhibits the transfer of prolineXY to sRNA, but does not inhibit the proline-dependent ATP-PPi exchange. Rather, a 3,4-dehydroproline-dependent ATP-PPi exchange catalyzed by the prolyl RNA synthetase can be demonstrated. A 4-hydroxy-L-proline-dependent ATP-PPi exchange is also catalyzed by the enzyme when high concentrations of this imino acid are employed. Hydroxyproline at relatively high concentrations is not an effective inhibitor of the transfer of proline-Cl4 to sRNA. Some of the properties and conditions for optimal activity of the purified enzyme are reported. INTRODUCTION
A great volume of evidence has accumulated that in the process of protein biosynthesis amino acyl RNA synthetases play an intermediary role by activating and transferring amino acids to specific soluble ribonucleic acid molecules. These enzymes have been shown to catalyze an amino acid-dependent adenosine triphosphate-inorganic pyrophosphate exchange, suggesting the probable formation of amino acyl adenylateenzyme complexes. Further, in experiments employing U4-labeled amino acids, these enzymes have been shown to catalyze the formation of amino acyl RNA complexes (1). Several amino acyl RNA synthetases have been purified and studied (2-5); however, other than evidence for its existence (6), no detailed investigation has been made on a prolyl RXA synthetase.2 1 This work was supported in part by a research grant from the Robert A. Welch Foundation of Texas. 2 Subsequent to the preparation of this manuscript, a paper was published relating to the partial purification of a prolyl RNA synthetase from
In the course of an investigation of some aspects of proline metabolism in certain microorganisms, it was found desirable to study the nature of the reaction catalyzing the activation of proline for its subsequent incorporation into protein. It was therefore undertaken to purify and study the properties of the prolyl RNA synthetase from Escherichia coli, and to test the effect(s) of 3,4-dehydroproline (7)) a known proline antagonist in certain microorganisms, on the activity of the purified enzyme. Herein are reported the results of this investigation. MATERIALS
AND
METHODS
MATERIALS Soluble ribonucleic acid from E. coli was obtained from General Biochemicals, Chagrin Falls, Ohio. uL-ProlinecarboxyllC’4 was obtained from California Corporation for Biochemical Research. L-Proline-U-Cl4 was obtained from Nuclear Chicago Corporation. Calcium phosphate gel was prepared by a previously described procedure (8). rat liver. The enzyme from rat liver differs in several aspects from the enzyme reported herein (64.
147
148
NORTON
The DEAE-cellulose, Type 20, was obtained from Brown and Company. Potassium P3aP? was prepared by pyrolysis of potassium P? for 1 hour, and appropriately diluted samples were used without further purification. DL-3,4-Dehydroproline was kindly supplied by Drs. Robertson and Witkop, National Institutes of Health. The author is deeply greatful to Mr. Morris Key andMrs. Sandra Scholes for their capable technical assistance during the course of this work. ENZYME
ASSAYS
For the measurement of proline-dependent ATP-PPi exchange the complete reaction mixtures contained in 1 ml: L-proline, 5rmoles; Tris buffer, pH 7.5, 100 Fmoles; potassium fluoride, 50 pmoles; sodium pyrophosphate-Pa*, 1 rmole (lOO,OOO800,060 cpm); and an appropriate amount of the enzyme fraction. Blanks containing no proline were included in each assay, and the exchange activity due to the presence of proline was obtained by difference. The reaction mixtures were incubated for 10 minutes at 37”C, and the reactions were terminated by the addition of 0.5 ml of 7% perchloric acid. After the ATP had been adsorbed and eluted from acid-washed charcoal by the method of Berg (2), aliquots were plated on concentric ring planchets and dried. Radioactivity was measured by use of a Nuclear Measurements Corporation Proportional Counter. After correction for background radioactivity the percentage and rate of exchange were calculated by the method of Davie et al. (9). The specific activity of the enzyme is reported as the number of micromoles of pyrophosphate exchanged per milligram of protein per hour. For the assay of the formation of prolyl RNA the complete reaction mixtures contained in 0.5 ml: L-proline-U-CY4, 6 mrmoles (320,009 cpm), or nn-proline-carboxyl-Cr4, 35 mpmoles (220,000 cpm); sRNA (excess), 1.5 mg; potassium ATP, 0.4 pmoles; magnesium chloride, 10 Fmoles; potassium phosphate buffer, pH 6.8, 50 rmoles; and a rate-limiting amount of the enzyme fraction. The reaction mixtures were incubated for 10 minutes at 37”C, and the reactions were terminated by the addition of 3 ml of cold 67% ethanol containing 0.5 M NaCl. Washing of the sRNA precipitate was fashioned after the procedure of Berg et al. (10). The washed sRNA was then dissolved in 1 ml of water, a 0.5 ml aliquot was plated and dried, and the radioactivity was determined. When either sRNA or enzyme was omitted from the reaction mixtures, the radioactive levels were only slightly above that of background. The specific activity of the enzyme is reported as the number of millimicromoles of L-prolyl-RNA formed per milligram of protein per hour.
Protein concentrations of the cell-free extracts were determined spectrophotometrically by the method of Warburg and Christian (11). Crude cell-free extracts (sonic extracts and ammonium sulfate fractions) were always dialyzed prior to assay against 0.02M potassium phosphate, pH 6.8, containing 0.002M magnesium chloride and 0.01 iVl p-mercaptoethanol. PREPARATION
OF CELL
EXTRACTS
Cells of E. coli 9723, grown in 5 ml of saltsglucose medium (12) for 14 hours at 37”C, were used to inoculate 100 ml. of a sterile medium which contained; KHzP04 , 17.0 gm; KzHPOa , 21.8 gm; yeast extract, 10 gm; and glucose, 10 gm per liter. This culture, after incubation for 8 hours at 37”C, was used t.o inoculate 6 liters of the above enriched medium. The culture was then incubated for 18 to 20 hours at 37°C and was harvested with a Sharples centrifuge. Usually 20 to 25 gm wet weight of cells were obtained under the above conditions. The cells were then washed and resuspended in 65 to 70 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.002 M magnesium chloride and 0.01 M fi-mercaptoethanol. This suspension in 25 ml. portions was then exposed to sonic oscillation for 3 minutes using a Branson Sonifier. Undisrupted cells and cell debris were removed by centrifugation at 30,000 g for 20 minutes at 4°C. The sonic extract was stored at 4”C, and all subsequent operations were conducted at 0” to 4°C. PURIFICATION
OF PROLYL
RNA SYNTHETASE
To 58 ml. of sonic extract containing 18.2 mg protein per milliter was added 5.8 ml of a 10% solution of streptomycin sulfate. After stirring 15 minutes, insoluble material was removed by centrifugation. To the supernatant solution (58 ml) a saturated solution of ammonium sulfate was slowly added to give a solution 28% saturated with respect to ammonium sulfate. After stirring for 15 minutes the precipitated protein was removed by centrifugation at 20,OOOg. The residue was discarded and the supernatant solution was then made 45y0 saturated by addition of saturated ammonium sulfate solution. After stirring for 15 minutes the insoluble protein was removed by centrifugation as previously and dissolved in 15 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.902 M magnesium chloride and 0.01 M p-mercaptoethanol. This ammonium sulfate fraction was then dialyzed overnight in 2 liters of the same buffer to give 19.5 ml of solution containing 14.1 mg protein per milliliter. The dialyzed 28 to 45oj, ammonium sulfate fraction (18 ml) was then treated with 88 mg of calcium phosphate gel (0.35 mg of gel per milligram
PROLYL
RNA
SYNTHETASE
149
rate of a proline-dependent ATP-PPi exchange. In early studies using crude cellfree extracts, attempts were made to demonstrate a proline-dependent hydroxamate formation in the presence of hydroxylamine; however, no such formation could be shown. This was not surprising in view of findings that measurement of amino aciddependent hydroxamate formation is too insensitive for the assaying of most amino acyl RiSA synthetases (13). Due to high pyrophosphatase activity in the crude enzyme extracts, it was necessary to add potassium fluoride (which effectively inhibited this activity) to the reaction mixtures when the pyrophosphate exchange assay was employed. Virtually all the pyrophosphatase activity was separated from prolyl RNA synthetase activity by selective adsorption on and elution from calcium phosphate gel (Table I, step III). The crude extracts were always dialyzed overnight before assaying. Even after extensive dialysis the crude extracts catalyzed an appreciable prolineindependent ATP-PPi exchange. However, the stimulation of the exchange activity upon addition of proline to the reaction mixtures was both significant and consistent. As the proline-dependent ATP-PP i exchange activity was purified the proline-independent ATP-PPi exchange activity was greatly decreased. As summarized in Table I, a purification of the prolyl RNA synthetase activity of approximately 20-fold has been obtained enzyme purification using conventional techniques. Enzyme fractions at each stage of purification were assayed by both prolinedependent ATP-PP i exchange and transfer of proline-Cl4 to sRNA. Both assay techniques gave good agreement with respect to the relative increase of enzyme specific activities at each purification step. In order that there be no doubt that the enzyme under investigation is actually a proline-activating enzyme, both DL-prolinecarboxyl-Cl4 and L-proline-U-Cl4 (obtained RESULTS AND DISCUSSION from different sources) were tested in assays For the assay for the presence of a prolyl involving transfer to sRT\‘A. Calculations of RNA synthetase in extracts of E. coli 9723, enzyme-specific activities were the same two procedures were employed; namely, the regardless of which proline substrate was measurement of transfer of CY4-labeled pro- employed. Further evidence was obtained line to sRNA and the measurement of the by use of paper chromatography. Enzyof protein). The pH was adjusted to 6.0 with 1 N acetic acid, the slurry was stirred for 15 minutes, and the gel was separated by centrifugation. Most of the desired enzyme activity was adsorbed on the gel by this procedure. The gel was washed by stirring with 20 ml of 0.02 M potassium phosphate buffer, pH 6.8, containing 0.002 M magnesium chloride and 0.01 M p-mercaptoethanol, and removed by centrifugation. The desired enzyme activity was eluted from the gel by stirring 15 minutes with 20 ml of 0.03 M potassium phosphate, pH 7.5, containing 0.002 M magnesium chloride and 0.01 M fl-mercaptoethanol. The gel was removed by centrifugation, and the supernatant solution (20 ml containing 1.8 mg protein per milliliter) was recovered. Most of 6he desired enzyme activity was eluted at this stage. Further elution with more concentrated buffer solutions failed to yield additional enzyme activity. Further purification of the prolyl RNA synthetase was obtained by use of a DEAE-cellulose column. The calcium phosphate gel eluate (15 ml) was passed through a column 1 cm in diameter containing 250 mg of DEAE-cellulose that had been previously washed with dilute hydrochloric acid and dilute sodium hydroxide and equilibrated with 0.03 M potassium phosphate, pH 7.5, containing 0.01 M fl-mercaptoethanol and 0.002 M magnesium chloride. After the gel eluate had passed through, the column was washed with 10 ml. of the same buffer. The prolyl RNA synthetase activity was recovered by successive additions to the column of 0.05, 0.07, 0.08, 0.09, 0.12, and 0.15 M potassium phosphate buffers, pH 7.5, containing the usual concentrations of P-mercaptoethanol and magnesium chloride. Fractions of 5 to 8 ml were collected and assayed. The desired enzyme activity was eluted from the column at buffer concentrations of 0.07 to 0.09 M. Active fractions were combined to yield 20 ml of solution containing 0.2 mg of protein per milliliter. The purified enzyme loses only a small amount of catalytic activity on storage at 4” C for one month, while complete loss of activity results upon exposure to a temperature of 60” C for a 5 minute period. Reducing conditions are necessary to prevent loss of prolyl RNA synthetase activity; therefore, the enzyme solutions were maintained in the presence of fi-mercaptoethanol throughout the purification procedure.
150
NORTON
PURIFICATION
OF
TABLE
I
PROLYL
RNA
SYNTHETASE Specific
Enzyme
fraction
I. Sonic extract II. Ammonium sulfate fraction 28-45% III. Calcium phosphate gel eluate IV. DEAE-cellulose
Yield (%I
100 60
Transfer to sRNAa (mpmoles/ Ww protein)
1.5 3.8
Activity ATP-PPi exchange* (pm&s/ hr/w protein)
0.08 0.20
25
10
0.40
10
30
1.8
a The reaction mixture contained: nn-proline, 35 mmoles, 2.2 X lo6 cpm; sRNA, 1.5 mg; potassium ATP, 0.4 pmole; magnesium chloride, 10 rmoles; potassium phosphate, pH 6.8, 50 pmoles; and a rate-limiting amount of each enzyme fraction. The reaction mixture volume was 0.5 ml and incubation was for 10 minutes at 37”. * The reaction mixture contained: L-proline, 5 pmoles; potassium ATP, 3 pmoles; magnesium chloride, 10rmoles; Tris buffer, pH 7.5,lOOrmoles; potassium fluoride, 50 pmoles; sodium pyrophosphate-Paz, 1 pmole, 8 X lo5 cpm; and a rate-limiting amount of each enzyme fraction. The reaction mixture volume was 1.0 ml and incubation was for 10 minutes at 37”.
matically produced Cr4-labeled sRNA was subjected to mild alkaline hydrolysis at 37”C, and the sRNA was then precipitated with ethanol. After concentration, the superC14-labeled natant solution (containing amino acid) was chromatographed on paper along with unlabeled n-proline in several solvents. In each instance the Rf value of the radioactive spot corresponded to that of the unlabeled proline. Some of the conditions for optimal activity of the prolyl RNA synthetase were studied. Variation of the pH between 6.8 and 7.6 had little effect on enzyme activity with either the exchange or the transfer reactions. Tris buffer, pH 7.5, was used with the pyrophosphate exchange assay, since phosphate buffer caused some precipitation in the reaction mixtures. Potassium phosphate buffer, pH 6.8, was employed with the transfer assay since there was the possibility of some hydrolysis of the enzy-
matically produced prolyl RNA at higher pH values. Reaction rates were linear with time for 10 minutes with both assay procedures. The effect of increasing concentrations of ATP on the formation of CY4-prolyl RNA was determined. From a LineweaverBurk plot of the data obtained (Fig. 1) the apparent K, value for ATP was found to be 6 X 1O-4 M. Concentrations of ATP greater than 0.6 pmoles per milliliter inhibited the transfer. It has previously been reported (7) that DL-3 ,4-dehydroproline inhibits the growth of several microorganisms including E. coli 9723, and that the toxicity is reversed in a competitive manner by n-proline. It was therefore of interest to test the effects of this inhibitor on the activity of the partially purified prolyl R3A synthetase, since the inhibition of proline activation would certainly result in cessation of cellular growth. As shown in Fig. 2, DL-3 ,4-dehydroproline
0
I
2
3
4
:
I/lSl x 10-S VATP, moles per liter FIG. 1. Lineweaver-Burk plot of the effect of ATP concentration on the transfer of n-proline-Cl” to sRNA. Reaction velocity (v) is in terms of moles per liter of prolyl RNA formed per second. The reaction mixture contained in a volume of 0.5 ml: L-proline-U-C14, 6 mpmoles, 3.2 X lo5 cpm; sRNA, 1.5 mg; magnesium chloride, 10 Imoles; potassium phosphate, pH 6.8, 50 rmoles; enzyme (DEAEcellulose fraction), 66 rg; and potassium ATP, 0.1 to 0.6 rmoles. Incubation was 10 minutes at 37°C.
PROLYL
RNA
inhibits prolyl RNA synthetase activity in a competitive manner. The apparent K, value for L-proline was calculated and found to be 1.2 X 1O-4 M. The apparent Ki value for 3,4-dehydroproline (based on the amount of ~-3 ,Pdehydroproline present) was calculated to be 12 X 10h4M. Experiments similar to that shown in Fig. 2 were run’ several times, and in each case the inhibition exhibited by the analog was competitive with proline. Thus in the intact microorganism, it is quite probable that at least one of the sites of inhibition of proline metabolism by DL-3,4-dehydroproline is at the site of proline activation. The transfer of amino acids to sRn’A molecules is a two-step process, the first being the formation of an amino acyl ade-
151
SYNTHETASE TABLE
16-
AND
EXRNA
Racii;.ci;ity Ra$mc$tity
None L-Praline 5pmoles L-Pro,ine’ 5 rmo,es + n;-3,4-dehydroproline, fimoles nL-3,4-Dehydroproline, 5 rmoles
(cpm)
(corrected for no praline control) (cpm)
875 3020 2905
0 2145 2030
1575
700
2940
2065
2820
1945
1190
315
1520
645
15
DL-3,4-Dehydroproline, 15 rmoles L-Proline, 5 rmoles 4-hydroxy-Lf proline, 20 Mmoles 4-Hydroxy-L-proline, 10 .umoles 4-Hydroxy-L-proline, 20 pmoles
18-
II
THE EFFECTS OF DL-3,kDEHYDROPROLINE 4-HYDROXY-L-PROLINE ON THE ATP-PPi CHANGE CATALYZED BY PURIFIED PROLYL SYNTHETASE
a The reaction mixture contained in 1 ml.: potassium ATP, 3 pmoles; magnesium chloride, 10 rmoles; Tris buffer, pH 7.5, 100 pmoles ; sodium pyrophosphate-P32 4.6 X lo5 cpm, 1 pmole; and enzyme (DEAE-cellulose fraction), 30 ,q. Incubation was for 10 minutes at 37°C.
Km for Proline
=
1234567 I/K1 x to-4 I/ Proline,
moles per liter
FIG. 2. Lineweaver-Burk plot showing the competitive inhibition of prolyl RNA synthetase activity by DL-3,4-dehydroproline. Conditions were as described under Fig. 1 except that 0.4 Fmoles of potassium ATP and 15 fig of enzyme (DEAE-cellulose fraction) were employed. The inhibitor concentration was 0.35 rmoles per 0.5 ml. O-O, L-proline; O-O, L-proline $ DL-3,4dehydroproline.
nylate-enzyme complex, the second being the transfer of the activated amino acid to a specific sRNA molecule. Inhibition of the over-all process can occur at either of these intermediate reaction processes (1). It was of interest, therefore, to determine whether the prolyi RNA sykthetase is capable of catalyzing a 3,4-dehydroproline-dependent ATP-PPi exchange or whether the exchange reaction is inhibited by the presence of this analog. As shown in TabIe II, DL-3,4-dehydroproline has no significant inhibitory action on the proline-dependent ATP-PPi exchange, while the analog does itself promote an ATP-PPi exchange. It has been reported (14) that in E. coli exogenously supplied 3,4-dehydroproline is incorporated into abnormal protein molecules in place of proline. Results obtained in this study are
NORTON
compatible with this finding. It is possible that 3,4-dehydroproline is transferred to the appropriate sRNA molecule as is L-proline, and the inhibition of transfer of L-proline-Cl4 to sRNA exerted by the analog (Fig. 2) is the result of its competition with L-proline for activation by the enzyme. As seen in Table II, 4-hydroxy-L-proline at high concentration also promotes an ATP-PPi exchange catalyzed by the prolyl RNA synthetase. Hydroxyproline in the presence of proline has no significant effect on the exchange reaction. In other experiments, hydroxyproline, at concentrations as high as 4 Itmoles per milliliter of reaction mixture, was found to have no significant effect on the transfer of proline-Cl4 to sRNA. It is probable that very high concentrations of hydroxyproline would be required to inhibit the normal catalytic function of the prolyl RNA synthetase of E. coli 9723. REFERENCES 1. BERG, P., Ann. Rev. Biochem. 30, 293 (1961). 2. BERG, P., J. Biol. Chem. 222, 1025 (1956). 3. SCHWEET, R. S., AND ALLEN, E. H., J. Biol. Chem. 233, 1104 (1958).
4. BERGMANN, F. H., BERG, P., AND DIECKMANN, M., J. Biol. Chem. 236,1735 (1961). 5. NORTON, S. J., RAVEL, J. M., LEE, C., AND SHIVE, W., J. Biol. Chem. 236, 269 (1963). 6. PETERKOFSKY, B., AND UDENFRIEND, S., Federation Proc. 21, 169 (1962). 6a. FRASER, M. J., AND KLASS, D. B., Can. J. Biochem. Physiol. 41, 2123 (1963). 7. SMITH, L. C., RAVEL, J. M., SKINNER, C. G., AND SHIVE, W., drch. Biochem. Biophys. 99, 60 (1962). 8. WOOD, W. A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 214. Academic Press, New York, 1955. 9. DAVIE, E. W., KONIGSBERGER, V. V., AND LIPMANN, F., Arch. Biochem. Biophys. 66,21 (1956). 10. BERG, P., BERGMANN, F. H., OFENGAND, E. J., AND DIECKMANN, M., J. Biol. Chem. 236, 1726 (1961). 11. WARBURG, 0.: AND CHRISTIAN, W., Biochem. 2. 310, 384 (1941). 12. ANDERSON, E. H., Proc. Natl. Acacl. Sci. U. S. 32, 120 (1946). 13. LOFTFIELD, R. B., AND EIGNER, E. A., Biochim. Biophys. Acta 72, 372 (1963). 14. FOWDEN, L., NESLE, S., AND TRISTR.~M, H., Nature 199, 35 (1963).