two types of binding of erythromycin to ribosomes …quantities of erythromycin, and is therefore...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 3, Issue of February 10,pp. 727-735,1969

Printed in U.S.A.

Two Types of Binding of Erythromycin to Ribosomes from

Antibiotic-sensitive and -resistant Bacillus subtilis 168”

(Received for publication, August 16, 1968)

NANCY L. OLEINICK$ AND JOHK TV. CORCORAN$

From the Department of BiochemistI-y, Case Western Reserve University School of Medicine, Cleveland, Ohio &I06

SUMMARY

The reversible binding of erythromycin to ribosomes of Bacillus subtilis 168 has been studied with two bacterial strains, one sensitive to low levels of the antibiotic and the other a relatively resistant mutant. Each ribosome from the sensitive strain binds approximately 1 molecule of erythromycin when a concentration of 4 x 1OV M is reached. As the concentration of antibiotic is raised to lo-” M no further binding takes place. At still higher concentrations, multiple interactions of erythromycin and the ribosomes occur. In contrast to the 1st molecule of erythromycin, which is specifically bound to the 50 S ribosomal subunit, the additional antibiotic molecules are bound nonspecifically to either the 30 S or 50 S particles. When resistant ribosomes are studied, a single molecule of erythromycin can bind to each 50 S subunit, so long as the concentration of antibiotic is kept below 10-j M. At higher concentrations, multiple interactions can take place, as with sensitive ribosomes. The main difference between sensitive and resistant ribosomes is that, with the former, the 1st erythromycin molecule is bound with sufficiently high affinity to produce a discontinuity in the curve relating the amount bound and the concentration. The 1st molecule of antibiotic is bound to each type of ribosome in the concentration range believed to exist within the cell when multiplication (protein synthesis) is inhibited. With either sensitive or resistant ribosomes, it appears that it is the 1st bound molecule of antibiotic which affects bacterial protein synthesis. Measurements of erythromycin binding under equilibrium conditions have revealed association constants of 2.6 X lo7 and approximately 8 X lo5 M-' for sensitive and resistant ribosomes,

* This work was supported by Research Grants AI-06758, 5.Tl-6M-35, and GM-AM 13971 from the United States Public Health Service and Grant 65G126 from the American Heart Asso- ciation. A portion of this material was presented at the 51st Annual Meeting of the Federation of American Societies for Ex- perimental Biology in Chicago, April 1967 (OLEINICK, N. L., AND CORCORAN, J. W., Fed. Proc., 26, 285 (1967)).

f Postdoctoral Fellow of the National Institutes of Health, United States Public Health Service.

0 Career Development. Awardee 5-K3-6M-2545, United States Public Health Service. Present, address, Department of Rio- chemistry, Northwestern University School of Medicine, Chicago, Illinois 60611.

respectively. Thus the mutant B. subtilis is probably resistant to erythromycin because of some change in ribosomal structure resulting in a lowered binding affinity for this antibiotic.

Binding of the 1st molecule of erythromycin to sensitive ribosomes is dependent on the presence of monovalent cations (40 mM NHdt or Kf). The bound erythromycin is freely exchangeable with erythromycin of the medium, and the binding is unaffected by the presence of tetracycline, chlortetracycline, or chloramphenicol. Implications re - garding the identity of the site (donor or receptor) occupied by erythromycin are discussed.

Erythromycin A (erythromycin) is a medium ring macrolide

antibiotic which inhibits the transfer of amino acids from amino-

acyl-tRNA into polypeptide linkage (I). Studies in our labora- t,ory on the mechanism of this inhibition have been aimed at correlating the physical interaction of erythromycin with the

bacterial apparatus required for protein synthesis and the resultant inhibition of this process. For this purpose, we have utilized ribosomes and enzyme fractions from either of twcJ strains derived from Bacillus subtilis 168; one, the sensitive strain, is inhibited by concentrations of erythromycin approxi-

mately 100-fold lower than those required to inhibit the other, a relatively resistant strain (2, 3). Polypeptide synthesis in cell- free systems derived from these two strains shows a 15- to 20- fold difference in sensitivity to erythromycin, and this sensitivity is determined to a major extent by the 50 S ribosomal subunit (4). These findings are of interest in light of the earlier results of Taubman et al. (5) which showed that tritium-labeled erythromycin is bound to the 50 S ribosomal subunit from the sensitive strain of B. subtilis under conditions in which binding to the 50 S subunit from the resistant strain, or to either type of 30 S subunit, is not’ evident. I f the bound antibiotic is required for

1 The terms sensitive and resistant are applied to the two strains of Bacillus subtilis that are, respectively, sensitive or resistant to the action of erythromycin A and to cellular components isolated from these strains. The quantity of ribosomal material is ex- pressed as ~4260 units.

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72s Erythl-omycin Binding to B. subtilis Ribosomes Vol. 244, No. 3

any inhibition of protein synthesis, it appeared that at higher erythromycin concentrations it should be possible to show binding to resistant ribosomes. In addition, there should be some definite relationship between the mole fraction of ribosomes with bound antibiotic and the resulting inhibition of polypeptide synthesis. A role of bound erythromycin in the inhibition of polylysine synthesis has been suggesetd by the results of Tanaka and Teraoka (6).

The experiments reported in this communication show that erythromycin binds to bot.h sensitive and resistant ribosomes from B. subtilis. At concentrations of probable pharmacological significance, a single molecule of antibiotic is bound per ribosome and this binding involves the 50 S subunit. The antibiotic sensitivity of polypeptide synthesis on sensitive and resistant ribosomes correlates well with the affinity of erythromycin for each type of ribosomes. Furthermore, it appears that inhibition of polypeptide synthesis in these systems is produced only by the bound erythromycin. The requirements for binding suggest that erythromycin interacts with the site normally occupied by polypeptidyl-tRNA.

EXPERIMEKTAL PROCEDURE

Preparation of 3H-Erythromycin

Streptomyces erythreus (strain CA340, Abbott Laboratories) was cultured as described by Kaneda et al. (7), and the washed mycelium was incubated for 8 hours with sodium propionate- 2,3-3H (5 mC, 238 or 410 mC per mmole).

The mycelium-free supernatant solution was diluted 2-fold with water to reduce the phosphate concentration and passed through a column (2 x 10 cm) of Amberlite resin IRC-50 which had been equilibrated with potassium phosphate buffer (1 InM,

pH 7.0) containing EDTA (1 mar). The column was washed with 200 to 300 ml of the same buffer, and the erythromycins were finally eluted with 1 M buffer. Ten-milliliter fractions were collected. Peaks of radioactivity were found at Frac- tions 4 to 5, 7 to 8, and 12 to 15. The third peak routinely contained more than 8O70 of the eluted radioactivity and all of the biologically active material (2). The identities of the other materials are unknown.

The erythromycin-containing fractions were pooled and purified in one of two manners. In early preparations, the material was ext,racted into methylisobutylketone by a five- chamber countercurrent distribution procedure with 5 X 70 ml of the organic solvent and 4 x 5 ml of distilled water. Five 250.ml reagent bottles were used, and each lower aqueous layer was transferred to the succeeding bottle by syringe. Of the radioactivity, 90 to 957, could be extracted. The extracts were pooled, and the methylisobutylketone was removed under reduced pressure. The residue was dissolved in acetone and subjected to partition paper chromatography by the method of Friedman, Kaneda, and Corcoran (8), with the upper phase of System A (n-heptane, benzene, acetone, isopropyl alcohol, 10 m&r potassium phosphate, pH 7.0, 25:50: 15: 10:25). The chromatogram was cut lengthwise into 3- to 4-cm strips for independent identification of radioactivity in a paper strip counter and comparison against positions of standards which were detected by spraying with a vanillin-HC104 reagent (8). The radioactive area corresponding to erythromycin A was eluted with acetone. This material was homogeneous and corre- sponded t,o standard erythromycin A as characterized by paper

chromatography with System B (methylcyclohexane, methylisobutylketone, t-butyl alcohol, 10 mM potassium phosphate, pH 7.0, 75: 10: 15: 100) (8). Other radioactive areas on the first chromatogram included small amounts of biologically active erythromycin B-like material and the relatively inactive erythromgcin-spiroketal, in addition to several small areas near the origin, all of which were biologically inactive.

The maiu radioactive fraction (Peak 3) from the IRC-50 fractionation of later preparations was subjected to a modified purification scheme. The pH of the combined fractions was adjusted to 9.5 with 2 M KOH to convert all of the desosamine- containing materials into the uncharged form. These were extracted with 4 x 30 ml of methylcyclohexane. The combined organic layers, containing about 75% of the radioactivity, were washed with 10 0.5.ml aliquots of water in a 250.ml bottle. Each aliquot was removed by aspiration prior to addition of the next aliquot. As the pH of successive washes dropped, increasing quantities of erythromycin were found in the aqueous phase, which was subsequently adjusted to pH 7 and reduced to 0.5 ml under nitrogen. This material was fractionated by column partition chromatography as follows: 85 g of silica gel (SG-34, Whatman) were combined with 85 ml of the lower phase of System A (see above) and added slowly with tamping to a 2-cm wide column containing the upper phase of System A. FinaI bed height was 77 cm. The sample was applied in 1.0 ml of lower phase. Elution was continued with the upper phase of System A. Five-milliliter fractions were collected at a flow rate of 0.5 ml per min. Under these conditions, radioactive peaks emerged at approximately 140.ml, 225.ml, and 370-ml elution volumes. The third peak contained 75% of the eluted radioactivity and all of the biologically active material. The product was homogeneous and identical with standard erythromycin A on paper chromatography in Systems A and B. Both methods used yielded chromatographically pure erythromycin A. However, the latter method is simpler and applicable to larger quantities of erythromycin, and is therefore preferred.

The amount of erythromycin A obtained was determined by a turbidimetric bioassay with the sensit’ive strain of B. subtilis (2). Radioactivity was measured by the liquid scintillation method. The three preparations used in this study had specific activities of 20.6, 59.0, and 115 PC per pmole.

Preparation of B. subtilis Ribosomes, Subribosomal Particles, and Xupernatant Enzyme Fraction

Procedures for the separation of the components of the cell- free, polypeptide-synthesizing systems from sensitive and resistant B. subtilis have been described (4). Because of the known lability of the 30 S subunit from B. subtilis (9), extensive washing of the ribosomes was generally omitted. The results are es- sentially the same for washed and unwashed ribosomal preparations. Ribosomes were stored at -80” in Buffer A: Tris-HCl (10 mM, pH 7.6), magnesium acetate (12 InM), and ammonium chloride (40 InM).

Jfeasurement of Binding of 3H-Erythromycin to Ribosomes

Ethanol Precipitation of Ribosomes-Incubation mixtures were prepared in 12-ml glass conical centrifuge tubes and consisted of Tris-HCl buffer (10 mM, pH 7.6), varying concentrations of magnesium acetate, ammonium chloride, and 3H-erythromycin, as shown in the legends to t,he figures, and 2.5 Anti0 units of ribosomes in 0.25-ml total volume. The binding of erythromycin


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Issue of February 10, 19G9 N. L. Oleinick and J. W. Cal-coran 729

to ribosomes is very rapid even at 0”. Routinely, a 10. to 15- min incubation was performed, usually at 22-23”. Following the incubation, 2.5 ml of cold absolute ethanol containing 40 mM NH&l were added. The 3H-erythromycin-ribosome complex is stable to dilution with ethanol, as long as the concentration of NH,+ is not permitted to drop below 40 InM. The ribosomal precipitate was allowed to coagulate at -20” for at least 1 hour, and was then collected by low speed centrifugation (-2400 x g, 2 to 3 min). The resulting pellet was washed with another 1.5-ml portion of the ethanol-NH&l solution, so that unbound 3H-erythromycin trapped in the pellet was reduced to a negligible amount. The final pellet was dissolved in aqueous ammonia (0.2 ml, 1 M) and transferred to a scintillation visl. The tube was rinsed with an additional 0.2 ml of 1 M ammonia, which was added to the same scintillation vial. Results obtained by this method agree closely with those obtained under equilibrium conditions.

Sucrose Density Gradient Centrifugation-Incubation mixtures as described above were assayed for the binding of erythromycin to 70 S ribosomes and to 50 S and 30 S subunits by layering them over 4.4-ml linear sucrose density gradients (25 to 10%) prepared in a buffer mixture composed of Tris-HCI (10 mM, pH 7.6), and the same concentrations of magnesium acetate and ammonium chloride as used in the reaction mixtures. Gradients were centrifuged for 135 min at 35,000 rpm and 3” in the SW 39 rotor in a Beckman-Spinco model L2 ultracentrifuge. The tubes were then punctured with a needle and 15-drop fractions were collected from the bottom. Each fraction was diluted with 0.7 ml of Hz0 and its optical density was determined at 260 rnp, after which the entire fraction was transferred to a scintillation vial for counting of radioactivity.

dleasurement of Binding at Equilibrium-The equilibrium dialysis method for measuring the interaction of 3H-erythromycin with ribosomes was considered t’o be undesirable as a result of the questionable stability of the B. subtilis ribosome during the long periods necessary to assure attainment of equilibrium, and also because of the large amounts of 3H-erythromycin required. Accordingly, the method of Hayes and Velick (10) was modified for these particular experimental conditions. Reaction mixtures of 1 .O ml were prepared in 2.0-ml cellulose nitrate tubes (A x 1% inch, Spinco Catalogue 303369). These contained Tris-HCl (pH 7.6, 10 /Imoles), magnesium acetate (12 pmoles), ammonium chloride (40 pmoles), sensitive or resistant ribosomes (100 to 400 A2e0 units), and various concentrations of 3H-erythromycin (20.6 or 59.0 PC per pmole). After mixing, a 100-~1 aliquot was removed to a scintillation vial for an exact measure of the initial concentration of erythromycin [Einitial]. The remain- der of the mixture was incubated for 15 min at 0”. Longer incu- bations of up to 3 hours did not alter the amount bound. The tube was then centrifuged for 15 min at 40,000 rpm and O-5” in the Spinco No. 40 rotor in a Beckman Spinco model L2 ultracentrifuge. A second IOO-~1 aliquot was then removed from the top of the tube to another scintillation vial and the radioact,ivity determined as a measure of the concentration of free (unbound) erythromycin at equilibrium, [Efree].

A control tube containing ribosomes but no 3H-erythromycin was treated in the same manner as the experimental tubes. Following centrifugation, nine 100.~1 aliquots were removed one at a time from the top of the liquid and diluted to 1 .O ml in Buffer A, and the optical density was determined at 260 rnp. More than 99 y0 of the 260 rnp absorbing material was found in aliquots

from the lower quarter of the original incubation mixture. Therefore, it was unnecessary to correct the value of [Erreel for the presence of erythromycin-ribosome complexes in the 100~~1 aliquot removed from the top of the tube. A second control tube, which contained 3H-erythromycin but no ribosomes, was also treated in the same manner as the experimental tubes. After the centrifugation, nine 100.~1 aliquots were removed in intervals from the top of the liquid and counted. During the centrifugation, no gradient of free erythromycin was established, so that it was unnecessary to correct for loss of free erythromycin from the top of the tube due to its sedimentation.

The two values determined as described above were used to calculate the concentration of bound erythromycin at equilibrium as follows: [EbounJ = [Ei,iti.l] - [Erree]. The concentration of ribosomes containing n binding sites per ribosome is defined as [RI. This term was calculated from the RNA content of the ribosomes as measured by the orcinol method (II), the value of 1.32 as the ratio of purines to pyrimidines in B. subtilis ribosomes (12), and the assumption that these ribosomes contained 60% RNA with molecular weights of 0.56 and 1.1 x lo6 (13). The average number of moles of erythromycin bound per mole of ribosomes is [E,,ound]/[R] h’ h d fi d w 1c IS e ne as B. If all of the binding sites are equivalent and independent, then

Kdissociation = (dR1 - L%oundl) PLel

[.%oundl

and

y=n--krd&]

Therefore, in the ideal case, a plot of ?/[Efree] against P will give a straight line, the slope of which is -Kd and the intercept on the pi axis, n (14).

Other Methods

The incorporation of Y-lysine into polypeptides, directed by polyadenylic acid as messenger RNA, was measured as described by Wilhelm and Corcoran (4). Ribosomal RNA was extracted from sensitive and resistant ribosomes by the phenol-sodium dodecyl sulfate method of Kurland (13). Radioactive samples were counted in 10 ml of a dioxane-based scintillation fluid (15) in a Nuclear-Chicago Mark I liquid scintillation spectrometer, with the automatic external standard channels ratio method for measuring the degree of quenching.

Materials

Sodium propionate-2,3-3H was purchased from Nuclear- Chicago and 14C-lysine from New England Nuclear. Erythro- mycin ,4 was a gift of Abbott.

RESULTS

The stoichiometry of the interaction of erythromycin with both sensitive and resistant B. subtilis ribosomes has been determined by the ethanol precipitation assay. Fig. 1 presents results typical of a large number of experiments. Up to an antibiotic concentration of 4 x 10-T M, erythromycin is bound to sensitive ribosomes in increasing amount’. In t’he concentration range between 4 X 10-V M and 10m5 M, there is no further increase in the amount bound. This quantity corresponds to 0.8 molecule of erythromycin per ribosome. For a number of ribosome


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730 R’rythromycin Binding to B. subtilis Ribosomes Vol. 244, No. 3

160

140

120

log EoDC concentration(M)

FIG. 1. Binding of erythromycin to sensitive and resistant ribosomes as a function of erythromycin concentration. The standard incubat,ion mixtures contained in 0.5 ml: Tris-HCI (pH 7.6, 5pmoles), magnesium acetate (6 @moles), ammonium chloride (20 pmoles), the indicated concentration of erythromycin, con- sisting of combinations of 3H-erythromycin (115 PC per pmole) and unlabeled erythromycin, and ribosomes (4.8 Am units). Ribosomes were added last and the reactants were incubated at 23” for 10 min, after which the ribosomes were precipitated with ethanol as described under “Experimental Procedure.” El, sensitive ribosomes; i~i, resistant ribosomes. EaDC, erythromycin A.

1 I I A: 6:

1.2 X 10-4M y, 1.2 X lO-2 M M$+ I

0.4 -

T L 0.3-

.L.

2

z 0.2-

2

0.1 -

Mg++ ii

70s :,

‘1; 600

Fraction Number

FIG. 2. Sucrose density gradient analyses of erythromycin binding at two Mgz+ concentrations. Standard incubation mixtures contained in 0.25 ml: Tris-HCl (pH 7.6, 2.5 pmoles), ammonium chloride (10 pmoles), the indicated concentrations of magnesium acetate, 3H-ergthromgcin (5 mpmoles, 115 PC per Mm&e), and sensitive ribosomes (2.5 A260 units). The tubes w&e incubated at 23” for 10 min: 0.2 ml was then lavered on a 25 to 10% linear sucrose density gr&dient.

,- Centrifugation and analysis

were performed as described under “Experimental Procedure.” - - -, A260; --, counts per min.

preparations, the saturation level varied between 0.6 and 0.8, but the pattern was always similar. This level can be increased to 0.9 to 1.0 in the presence of puromycin, which removes nascent peptides from the ribosomes (16, 17), thereby presumably ex- posing more sites for erythromycin binding.2 Since the 1st

2 N. L. Oleinick and J. W. Corcoran, lunpublished observat,ion.

molecule binds to sensitive ribosomes at low concentrations, we refer to it as “high affinity” binding.

Bdditional binding (> 1 molecule per ribosome) takes place when the concentration of ant.ibiotic is raised above 10-j M. In some experiments, when the concentration was increased to 10V3 M, as many as 5 to 7 molecules were bound per ribosome. 9t concentrations above 10e5 M, the measurements are less reliable than at lower concentrations, since the specific activity of the added erythromycin is decreased considerably by the addition of unlabeled erythromycin. However, the general pattern of multiple binding above 10e5 M erythromycin was always found. When we repeated these measurements with resistant ribosomes, we found a different binding pattern. Less erythromycin is bound at the lower concentrations, and no saturation level is noticeable in the binding curve. At the higher antibiotic concentrations, the association curves for both sensitive and resistant ribosomes are identical, within the limits of the assay method.

The results of Taubman et al. (5) suggest that the high affinity binding of erythromycin to sensitive ribosomes occurs with the 50 S subunit. We have verified this by two different experiments. Fig. 2 illustrates two sucrose density gradient analyses of sensitive ribosomes incubated with 2 x lo-7 M erythromgcin, a concentration just below that which saturates the high affinity site. The incubation giving the results shown in Curve A was done at a low concentration of Mgzf, at which the free 50 S and 30 S subunits are preponderant. The distribution of erythromycin coincides with the distribution of the 50 S subunits, and little antibiotic is associated with the 30 S subunits. In the presence of a higher concentration of Mg2+, as shown in analysis Pattern B, the antibiotic is bound to both 50 S and 70 S particles.

Further verification that high affinity binding involves the 50 S subunit was obtained by measuring the interaction of erythromycin with separated 30 S and 50 S subribosomal particles from the sensitive B. subtilis (Fig. 3). It is clear that the 50 S subunit from the sensitive bacteria is capable of both multiple and high affinity binding. In contrast, t.he 30 S subunit binds antibiotic only at high concentrations. This is also true for the 30 S subunit from the resistant bacteria. Resistant 50 S subunits produce a binding curve similar in shape to those of either type of 30 S subunit., but more erythromycin is bound at any particular concentration. It is shown below that resistant 50 S subunits also possess a unique binding site for erythromycin, with an affinity lower than that of sensitive ribosomes.

The multiple binding of erythromycin to ribosomes might be due to a nonspecific interaction with RNA. This idea was tested (Fig. 4) by measuring the association of erythromycin with some synthetic polynucleotides (poly A,3 poly C, and poly U) and with ribosomal RNA extracted from both sensitive and resistant ribosomes by the method of Kurland (13). There is some interaction of the antibiotic with all of these RNAs, but only at high concentrations. No unique, high affinity binding is seen with any of these RN,4s, including that from the sensitive ribosomes. Results of a similar attempt to measure binding of 3H- erythromycin to a protein, bovine serum albumin, were negative.

It is conceivable that under some conditions erythromycin may be covalently bound t.o RNA or protein via Srhiff base- formation between the C-9 carbonyl group in the la&one portion

3 The abbreviations used are: poly A, pol\atlclrylic acid; poly C, polycytidylic acid; poly U, polyuridylic: aiid.


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Issue of February 10, 1969 N. L. Oleinick and J. W. Corcoran

Sensitive

-6 -5 -4

Resistant

731

log [EaDC (M I]

FIG. 3. Binding of erythromycin to subribosomal particles. Separated 50 S and 30 S subunits from sensitive and resistant ribosomes were incubated 10 min at 22” in 0.25-ml total volume in the presence of Tris-HCl (pII 7.6, 2.5pmoles), ammonium chloride (10 pmoles), magnesium acetat,e (3 pmoles), and the indicated concentrations of 3H-erythromycin (20.6 MC per pmole). The ethanol precipitation assay was used. 0, 50 S sllbllnit; q , 30 S subunit. EaDC, erythromycin A.

of the antibiotic and an amino group in the RSh or protein.

If so, such a bond should be susceptible to reduction to a stable

secondary amine by sodium borohydride. To test t,his possi-

bility, we incubated mixtures of 3H-erythromycin and sensitive

ribosomes for 15 min at 23”, and then treated them with XaRH1

and isolated separately the ribosomal RNA and protein (18). No erythromycin was associated with either component. Hence,

formation of a Schiff base between erythromycin and the ribo-

some does not occur in sufficient concentration to be detected by this method.

The binding of erythromycin to 50 S subribosomal particles from the sensitive strain of B. subtilis appears to be almost com-

pletely reversible (Table I). The 50 S subunits w-ere incubated with 3H- or unlabeled erythromycin, at 10m6 M, a concentration

which saturates the high affinity site, and then diluted with an equal volume of lo-” M 3H- or unlabeled erythromycin. The

amount of radioactivity bound to the ribosomes in each case is

nearly identical with that calculated assuming dilution of the

total 3H-erythromycin by total unlabeled erythromycin, and this

indicates that bound erythromycin can exchange with unbound

antibiotic in the medium.

The binding of erythromycin to the high affinity site on sensi-

tive ribosomes appears to be dependent on the concentrations of

both the monovalent and divalent cation (Fig. 5). In the

presence of high concentrations of Mg2+ (12 IIIM), the concentration of NH,+ is critical: almost no erythromycin is bound at 1 nm NH,+, and maximum binding is observed between 40 rnnl

and 1 M NH4+. On the other hand, when low concentrations of

Mg2f (0.12 mM) are present, there is a much less rigid require- ment for NHd+. Regardless of t’he Mg2f concentration used, t’he same level of binding is reached when the NH4+ concentration is

sufficiently high. On the other hand, there is little effect of

NH*+ concentration on the multiple binding of erythromycin to sensitive or resistant ribosomes. Multiple binding takes place

at very low concentrations of NH.++ (3 moles per mole of ribosome at 1 mhf NHh+). An additional 0.6 mole of erythromycin is bound when the NH4+ concentration is raised to 40 mM. This

NH4+-dependent increase is no more than that seen for high affinity binding alone. In the presence of 12 mM Mg2+, K+ will

replace NH4+ with about one-third the efficiency, while Na+ does not aid the binding of erythromycin.

In another communication (3) we have described the effect of a series of macrolide antibiotics, erythromgcin analogues, and lincomycin on eryt.hromycin binding, cell-free polypeptide synthesis, and growth of B. subtilis. All of the macrolides and

2 n p 40

i2 ResIstant r RNA / I

Sensitive r RNA

10-7 10-6 10-5 10-4

EoDC concentratbon (M)

FIG. 4. Binding of erythromycin to RNA. Standard reaction mixtllres contained in 0.25 ml: Tris-HCl (pH 7.6, 2.5 pmoles) ammonium chloride (10 pmoles), magnesium acetate (3 rmoles), the indicated concentrations of 3H-erythromycin (20.6 & per pmole), and 50 pg of poly A, poly C, or poly U, or 69 pg of RNA from sensitive ribosomes, or 65.3 rg of RNA from resistant ribosomes. Incubat,ion was for 10 min at 23”. The ethanol precipitation assay was used. 0, poly A; q , poly C; A, poly U; W, sensitive rRNA; l , resistanl rRNA. EaDC, erythromycin A.

TAHLE I

Reversibility of 3H-erythromycin binding to Bacillus subtilis ribosomes

Three incubation mixtures were prepared, each containing (in 0.5 ml) 1.37 112~~ units of sensitive 50 S ribosomes and 10-e M

erythromycin, either 3H-labeled or unlabeled as indicated, in Buffer A. After a 15.min incubation at 23”, each mixture was diluted with an equal volume of Buffer A containing 10-G M

erythromycin, either labeled or unlabeled as indicated. Follow- ing another 30.min incubation a,t 23”, the ribosomes were precipitated with ethanol-O.04 M NH4Cl as described under “Experi-

mental Procedure.”

Type of erythromycin present I I

Original incubation mixture Dilution mixture

dpm/pmole dPm pWbOleS

1. 3H-Labeled. 3H-Labeled 131 4250 32.4 2. 3H-Labeled. Unlabeled 65.5 2240 34.2

3. Unlabeled.. 3H-Labeled 65.5 1880 28.7

a Assuming complete mixing of labeled and unlabeled, bound

and unbound erythromycin.


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732 Er!/thromycin Binding to B. subtilis Ribosomes Vol. 244, Ko. 3

-3 -2 -I 0

log [Concentration NH,+ CM)]

&CT. 5. Dependency of binding of erythromycin on Mg2+ nnd NHI+ concentrations. A suspension of sensitive ribosomes which had been stored at -80” in Buffer A was thawed, diluted in half with a buffer containing 0.01 M Tris-HCI, pH 7.6, and 0.12 rnM magnesium acetate, centrifuged for 2 hours at 178,000 X g, and resuspended in the buffer containing low Mg2f and no NH4+. Of these ribosomes, 2.44 A~60 units were incubated for 10 min at 23” with 2 X 10m7 M 3H-erythromycin (115 pC per pmole) in the presence of 0.01 nf Tris-HCI, pH 7.6, and the indicated concentrations of Mg2+ and NHd+ in a total volume of 0.25 ml. The ethanol precipitation assay was used. &DC, erythromycin A.

analogues which resemble erythromycin A in possessing an aminodeoxy sugar in glycosidic linkage to a macrolide lactone interfere with the binding of 3H-erythromycin and are cross- resistant with erythromycin, probably because they interact with the same site on the ribosome. Lincomycin, although not a macrolide antibiotic, probably also shares at least part of the macrolide-binding site on the ribosome (3, 19,20). On the other hand, compounds which do not compete with erythromycin, e.g. chloramphenicol (al), may not interact with the erythromycin- binding site. It was therefore of interest to determine what effect the tetracycline group of antibiotics has, since they bind to both 30 S and 50 S subunits (22, 23) and inhibit the binding of aminoacyl-tRNA to the ribosome (24-26) Table II shows that tetracycline and chlortetracycline, at concentrations which profoundly inhibit polylysine synthesis, are without effect on the binding of 3H-erythromycin. Hence, these antibiotics must interact with the ribosomes at separate loci. Furthermore, erythromycin binding is unaffected by the presence of various components of the cell-free amino acid-incorporating system. The data of Table III show that at all levels of erythromycin binding poly U and crude tRNA have no significant effect.

It, is important to establish the function of the various erythromycin-binding sites on sensitive and resistant ribosomes. Ac- cordingly, the correlation between bound antibiotic and the coincident inhibition of protein synthesis was studied. The results are shown as Fig. 6. The binding of erythromycin to the high affinity site on sensitive ribosomes is paralleled by the inhibition of polylysine synthesis. When the first site is saturated, about 0.75 molecule is bound per ribosome and inhibition of polypeptide synthesis is maximal (approximately 80%). A correlation also exists between binding and inhibition with re-

TABLE II

h’$ect of tetracycline and chlortetracycline on binding of 3H-erythromycin to sensitive Bacillus subtilis libosomes

For the measurement of inhibition of polylysine synthesis,

incubation mixtures contained in 0.25 ml: Tris-HCl (pH 7.6, 2.5 rmoles), magnesium acetate (3 pmoles), ammonium chloride (10 pmoles), mercaptoethanol (15 pmoles), ATP (0.5 pmole), phos-

phoenolpyruvate (1.25 pmoles), pyruvate kinase (12.5 pg), GTP (0.02 rmole), a mixture of 20 Y-amino acids minus lysine (2 mpmoles each), tRNA (50 rg), i4C-lysine (0.2 PC, 0.85 mrmole),

sensitive ribosomes (2.5 4 260 unitas), poly A (25 rg), enzyme fraction (2.0 pg) (4), and the indicated concentrations of erythromycin, tetracycline, and chlortetracycline. The mixtures were

incubated for 30 min at 37”, after which a 50.~1 aliquot was transferred to a 2-cm filter paper disc and washed in 5yc trichloracetic acid as described previously (4). For measurement of erythro-

mycin binding, incubation mixtures contained in 0.25 ml: Tris- HCl (pH 7.6, 2.5 pmoles), magnesium acetate (3 rmoles), ammonium chloride (10 pmoles), and the indicated concentrations of

tetracycline, chlortetracycline, and 3H-erythronlycin (59.0 PC per fimole) . These were assayed by precipitation with ethanol.

Antibiotic

-0

Tetracycline

Chlortetra-

cycline

Concentra- tion

M

10-o 64.1

10-b 84.5 99.3 100 10-d 100

10-s

10-S PO-4

Inhibition of polylysine Erythromycin bound synthesis at erythromycin at 3H-erythromycin

concentration of concentration of

0 2 x 10-r M 10-s Y

% -

85.2 100 100 100 100 I I

1 x 10-7 I 10-o Y

pnto1es

18.3 37.3

17.1 38.4 16.7 40.5 18.2 35.4

19.2 37.6 18.7 35.2

15.7 33.6

a Control incubation, binding of 3H-erythromycin in the absence of tetracycline and chlortetracycline.

TABLE III

E$ect of poly U and tRNA on binding of 3H-erythromycin to sensitive ribosomes

Incubation mixtures contained in 0.25 ml: Tris-HCl (pH 7.6,

2.5 rmoles), magnesium acetate (3 pmoles), ammonium chloride (10 pmoles), sensitive ribosomes (2.4 A260 units), poly U (50 pg), or tRNA (50 rg) where indicated, plus the concentrations of

3H-erythromycin (115 PC per pmole or less when diluted with unlabeled erythromycin) specified in the table. Incubation and ethanol precipitation assay were as described under “Experi- mental Procedure.”

Erythromycin concentration I-

I- M

2 x lo-’

4 x 10-T 1.2 x lo-6

4 x 10-c 1.1 x 10-b

4.1 x 10-S 1.2 x 10-d

Erythromycin bound after additions of

Xone / poly U / tRXA / p;l$zA+

0.50

0.60

0.64

0.71 1.18 2.13

moles/mole ribosome

0.42 0.58

0.67 O.G3 0.64 0.65 0.81 0.93

1.54 3.44 2.78

0.55

0.67 0.66 0.74

0.83 1.38 2.84


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Issue of February 10, 1969 N. L. Oleinick and J. W. Corcoran 733

sistant ribosomes, where binding of 0.6 to 0.7 molecule of erythromycin occurs at the same concentration where a maximal inhibition of protein synthesis (approximately 65%) is observed. Resistant ribosomes bind nearly as much erythromycin and can be inhibited to almost the same extent as sensitive ribosomes, but 20-fold higher concentrations of antibiotic are required. Fur- thermore, most of the inhibition of protein synthesis seems to result from the binding of the first molecule of erythromycin. No significant inhibition is caused by further association of the antibiotic with either sensitive or resistant ribosomes.

These results suggest that, like sensitive ribosomes, resistant ribosomes also possess a specific and unique binding site for the 1st molecule of erythromycin. If so, it has a reduced ability to bind the antibiotic. Accordingly, we attempted to measure the binding constants and number of sites under conditions of

I.6 Sensitive

1.4 -

Resistant

log [EaDC Concentration (Md

FIG. 0. Correlation of erythromycin-binding and inhibit,ion of polypeptide synthesis. The cllrves for binding of erythromycin to sensitive and resistant ribosomes are those of Fig. 1. For the inhibition of amino acid incorporation, the conditions were as described in Table II. EaDC, erythromycin A.

I 2 3 v

FIG. 7. Scatchard plot of equilibrium binding data for resistant ribosomes. Conditions are described in the Experimental sec- tion. The solid line at 5 < 1.0 is that calculated by the met,hod of least squares.

0 0.2 0.4 0.6 0.8 1.0

i7

FIG. 8. Scatchard plot of equilibrium binding data for sensitive ribosomcs. Conditions are described under “Experimental Yro- cedure.”

thermodynamic equilibrium. Fig. 7 shows a Scatchard plot (14) of data obtained for the binding of erythromycin to resistant 70 S ribosomes. These ribosomes appear t.o possess one site with an association constant of approximately 8 x lo5 M+ and at least three other sites with lower association const,ants. ,4 similar treat-

ment of data for sensitive 70 S ribosomes yields a Scatchard plot (Fig. 8) with an anomalous curvature at low concentrations of erythromycin. The same type of plot. is obtained for the binding of 3H-erythromycin to sensitive 50 S ribosomal subunits. Hence the anomalous shape cannot be attributed to dissociation of 70 S ribosomes into 50 S and 30 S subunits and their reasso- ciation. We could not calculate an association constant from this curve. However, we estimate that its value is -2.6 X 107 M-I, which is the free concentration of erythromycin at half- saturation. The data for resistant ribosomes (Fig. 7) have been treated assuming that there is a linear relationship between ii/ [Efree] and F, and a least squares analysis of all points for which P <l has yielded a line which extrapolates to approximately 1 mole per mole of ribosome. It is possible, however, that more reliable data at very low values of ‘P would reveal a curvature similar to that of Fig. 8.

DISCUSSION

Erythromycin has been shown to bind in two ways to ribosomes of B. subtilis (Fig. 1). At low concentrations, ribosomes from a sensitive strain bind approximately 1 molecule of erythromycin to the 50 S subunit (Figs. 2 and 3). A similar stoichiometry has been reported for erythromycin-binding to ribosomes from Staphylococcus aureus (27). Although the mechanism of this interaction is unknown, the available evidence favors a nonco- valent linkage. Bound erythromycin exchanges readily with free erythromycin in the medium (Table I) and is dependent on a high concentration of monovalent cations (Fig. 5). It is not


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734 Erythromycin Binding to B. subtilis Ribosomes Vol. 244, Xo. 3

removed by treat’ment of the complex with ethanol during t’he precipitation assay (see “Experimental Procedure”), or by ceu- trifugation through sucrose (Fig. 2), but only as long as the NH4+ concentration is maintained at 40 InM or higher. Finally, Schiff-base formation involving the C-9 carbonyl group of the erythromycin lactone and a primary amine on the ribosomal RN,4 or protein could not be detected.

At high concentrations ( > lop5 M erythromycin), ribosomes from both the sensitive and resistant strains of B. subtilis bind more than 1 molecule of erythromycin (Fig. 1). It is probable that the mechanism by which this interaction occurs differs from that for the 1st molecule bound, since little, if any, dependence on NH4+ concentration could be shown. At these higher antibiotic concentrations, erythromycin itself may be able to replace NH4+ as a monovalent cation. The multisite binding occurs with both subunits from either strain (Fig. 3) and with various RNhs (Fig. 4).4

Erythromycin appears t,o act at some stage in the transfer of activated amino acids into polypeptide linkage (1). In the presence of erythromycin, there is a decrease in synthesis of longer lysine oligomers and an increase in di- and trilysines (28). An increase in shorter phenylalanine oligopepbides at the expense of the longer pol~phen~lalaniiies is also found, although the inhibition requires higher concentrations of erythromycin than wit,h the polylysine syst,em.j Such results could be explained if erythromycin becomes bound to one of the sites on the 50 S subunit normally occupied by a struct,ural portion of amino acyl- or peptidyl-tRNh, and by so binding partially prevents the en- trance of that species into its site. Indeed, structural features and similarity iu mechanism of a series of macrolide antibiotics, erythromycin analogues, and liucomycin suggest that the carbo- hydrate portions of erythromycin may be au analogue of the AT- ribosyl portion of a ribonucleotide in tRNA (3). I f the translocation model (29) of protein biosynthesis is correct, at least two tRNS binding sites must exist on the ribosomes. According t,o this model, the peptide portion of peptidyl-tRNA in the “donor” site is transferred to the free a-amino group on the aminoacyl- tRNA in the “receptor” site (30). Reports on t,he properties of these sites are numerous (e.g., References 24, 25, and 31 to 39), but it is not, always certain which site is being studied, since various conditions allow binding of peptidyl-tRNA and amino acyl-tRN.4 to either site (40).

Recently, Pulkrabek and Rgchlik (41) found that the binding of the 1st molecule of 1ysylLtRNA to the poly h-ribosome complex takes place in the absence of monovalent cations, while NH4+ or Kf is necessary for the second lysyl-DRNA to bind, after which lysyl-1ysylLtRNA forms in the absence of soluble enzymes and GTP, and is presumably bound to the receptor site. At this point, monovalent cations are not necessary for maintaining the interaction of lysyl-1ysyLtRNA and the ribosome, nor are theyre- quired to bind polylysyl-tRNA to the polg A-ribosome complex (42). Therefore, the receptor (aminoacyl) site appears to be

4 After this paper was submitted, a report (AHMED, A., Hiochirtz. Hiophys. dcta, 166, 218 (1968)) appeared concerning the mechanism of action of spiramycin, a macrolide antibiotic related to erythro- mycirr, on ribosomes from sensitive and resistant strains of B. subtilis W23. Ahmed reports that sensitive and resistant ribosomes bind the same amount of spiramycin. However, this was in the presence of approximately 1.6 mM spiramycin, at which concentration multiple site nonphysiological binding is probably occurring.

5 J. PI/I. Wilhelm and .J. W. Corcorau, lulpublished observation.

NH4f- or K+-independent (33, 38, 41) and message-specific (37-39), while the donor (peptidyl) site is NH*+- or Kf-dependent (33, 38, 40) and nonspecific with respect to message (an exchange site) (35). illthough there is still some question (cf. Reference 33), tetracycline sensitivity probably resides at the receptor site (22-25, 40).

Present’ evidence allows us to speculate as to which, if either, site is occupied by erythromycin. Components of the cell-free, amino acid-polymerizing system do not seem to be inhibitory t,o erythromycin binding (Table III). Erythromycin does not iuhibit the binding of specific amino acyl-tRNA to the 30 S ribosomes, but it does inhibit the 50 S subunit-dependent increase in binding (43). Tetracycline and chlortetracycline, at concentrations capable of completely inhibiting poly A-directed synthesis of polylysine, have no effect on erythromycin binding (Table II). Furthermore, erythromycin binding is dependent on the presence of NH4+ or K+. This suggests that erythromycin becomes bound to some portion of the peptidyl-tRNA site. Several other observations are consistent with this hypothesis. (a) Bound and unbound erythromycin are freely exchangeable (Table I). (b) pH studies suggest that erythromycin is more active when in the uncharged form. This form would more easil! bind to the donor site which normally accepts an uncharged polypeptidyl-tRNA as opposed to an aminoacyl-tRNA bearing a charged cr-amino group (44). (c) Stripping of nascent peptides from the ribosome by puromycin increases the binding of erythro- m.ycin.2 Puromycin has been shown to remove only part of the nascent peptides, specifically those bound to the donor site (30). (d) Er)-t,hromycin inhibits the puromycin reaction (42, 45) but not in the presence of tetracycline (45). Therefore, since tetracycline blocks the receptor site and puromycin reacts only with tRNh-bound peptides already attached to the donor site, erythromycin appears to bind to the donor site, and may act b> inhibiting the translocation of peptidgl-tRNA from the receptor t,o the donor site.

Inhibition of polypeptide synthesis appears to be associated with only the 1st molecule of erythromycin bound per ribosome, and no further inhibition is coincident with the multiple sit,e binding (Fig. 6). Hence, if resistant ribosomes had been found to contaiu only the nonspecific, nonfunctional multiple sites, it would be necessary to postulate that the inhibition observed in Fig. 6 was a result of nonbound erythromycin. However, the erythromycin-binding site on resistant ribosomes is not lost al- together but, rather, as a result of the mutation, it has a reduced affinity for the antibiotic (Fig. 7). Furthermore, the calculated affinity constants for the two erythromycin-ribosome complexes are consistent with a direct relationship between binding and inhibition. These values (2.6 x lo7 M-~ for sensitive and approximately 8 x lo5 M-I for resistant ribosomes) differ by a factor of 33 which is similar to the ratio of the sensitivities of the two cell-free polypeptide-synthesizing systems to erythromycin

(4).

The complex shape of the Scatchard plot for binding of erythromycin to sensitive ribosomes (Fig. 8) requires further comment. The vertical straightline portion at B = 0.6 presumably represents saturation of the first site by erythromycin and is less than 1 mole per mole of ribosomes because many of these crude ribosomes contain nascent peptide chains which may block the erythromycin-binding site. Further binding is of the multisite variety, since the points at ii > 0.6 extrapolate to a value greater than 1. The curvature at’ low values of p is not without precedent.


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Issue of February 10, 1969 N. L. Oleinick and J. W. Comwan 73.5

Changeux, Gerhart, and Schachman (46) have observed a similar curvature for the binding of succinate to the native form of aspartate transcarbamylase (carbamyl phosphate : L-aspartat,e carbamyl transferase, EC 2.1.3.2)) indicative of cooperative homotropic interactions. The explanation of the curvature in the erythromycin-ribosome binding plot is not known.

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Nancy L. Oleinick and John W. Corcoran 168Bacillus subtilisand -resistant

Two Types of Binding of Erythromycin to Ribosomes from Antibiotic-sensitive

1969, 244:727-735.J. Biol. Chem.

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