Biochimica et Biophysica Acta, 420 (1976) 155-164 O Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands BBA 37232

A SPECIFIC CEPHALOSPORIN-BINDING PROTEIN OF CITROBACTER

FREUNDII HIROSHI OGAWARA

Department of Antibiotics, National Institute of Health, Kamiosaki-2-chome, Shinagawa-ku, Tokyo 141 (Japan) (Received July llth, 1975)

SUMMARY

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any fl-lactamase activity at substrate concentrations examined: 6 mM to 100 #M of penicillins and 12 mM to 100/~M of cephalosporins. 4. In gel filtration, [14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence of the binding protein, while penicillins did not quench the fluorescence. Association constants of cephalosporins were in the range of 0.8-12. l03 M -1, and one binding site was calculated for all cephalosporins tested.

INTRODUCTION

Roles of fl-lactamases (penicillin and cephalosporin amido-fl-lactamases, EC 3.5.2.6) in bacterial resistance have been extensively studied. However, some papers have thrown doubt on the idea that the main function of fl-lactamase in cells is to hydrolyze various penicillins and cephalosporins to protect the cells from the effect of these antibiotics and have proposed re-evaluation of its physiological role in relation to other components in bacterial cells [1-4]. Some fl-lactamases have a broad optimum pH vs activity profile [5] and a fllactamase obtained from Citrobacterfreundii is reported to show different optimum pH depending on whether penicillin or cephalosporin is the substrate [6]. Therefore, it is not unreasonable to imagine that types of binding or reaction of some fl-lactamases with penicillin are different from those with cephalosporin, or that the binding sites for penicillin and cephalosporin are located in different positions in the enzyme protein molecule. This paper describes the isolation of a cephalosporin-binding protein from C. freundii and some of its properties. This binding protein appears to bind to cephalosporins but not to penicillins.

156 MATERIALS AND METHODS

Chemicals. Beef pancreatic chymotrypsinogen A and ovalbumin were purchased from Mann Research Laboratories. Trypsin (EC 3.4.4.4) was purchased from Worthington Biochemical Co., pepsin (EC 3.4.4.1) from Sigma Co. and crystalline bovine serum albumin from Nutritional Biochemical Co. Benzylpenicillin was obtained from Takeda Pharmaceutical Co., ampicillin from Meiji Seika Co., cloxacillin, cephazoline and cephalosporin C from Fujisawa Pharmaceutical Co., methicillin from Banyu Pharmaceutical Co., cephalothin from Eli Lilly Co., cephaloridine from Glaxo Laboratories, and cephalexin and cephaloglycin from Shionogi Pharmaceutical Co. Sephadex G-75 (40-120#m) and Sepharose-4B were the product of Pharmacia Co. [~4C]Benzylpenicillin was purchased from Radiochemical Centre, England (38.2 mCi/mmol). Acrylamide and N,N'-methylenebisacrylamide were purchased from Seikagaku Kogyo Co. Growth of organisms. C. freundii GN346, kindly supplied by Professor S. Mitsuhashi, Gunma University, was grown at 35 °C under aeration and stirring in 3 1 of nutrient broth overnight and inoculated into 28 1 of the same medium. At 2 h cultivation, potassium benzylpenicillin was added (for the induction of/%lactamase) to a final concentration of 1 ~ and further incubated for 2 h. The culture was stopped and the cells were harvested at 0 °C using a Tomy Seiko continuous flow centrifuge at 16 000 x g (Model RS-18P, Tomy Seiko Co.). The pellet thus obtained was washed with 300 ml of 0.85~o NaC1 and was centrifuged at 0 °C for 20 min at 17 000 x g. Molecular weight determination. Trypsin (mol. wt 23 000), chymotrypsinogen A (25 700), pepsin (35 000), ovalbumin (43 000) and serum albumin (68 000) were used as molecular weight standards. Disc electrophoresis containing sodium dodecyl sulfate was carried out at room temperature by the method of Weber and Osborn [7]. Protein determination. Protein was determined by the method of Lowry et al. [8] using crystalline bovine serum albumin as a standard. fl-Lactamase activity, fl-Lactamase activity was determined iodometrically at pH 7.0 by the slightly modified [9, 10] method of Perret [11]. The direct spectrophotometric assay methods of O'Callaghan et al. [12] and Samuni [13] were employed for measurement at dilute substrate concentrations. Amino acid determination. Approximately 300 fig of the salt-free protein, which had been obtained by exhaustive dialysis, were hydrolyzed in 0.3 ml of constant boiling HCI at 110 °C for 24 and 41 h in a sealed evacuated tube. The amino acid analysis was performed by the Hitachi KLA-3A analyzer. Fluorescence spectrophotometry. Hitachi MPF-4 spectrophotofluorometer was employed. In all experiments, the excitation slit was 4 nm and the emission slit was 10 nm. Emission spectra were obtained by an excitation at 290 nm, and monitoring was done at the peak near 335 rim. Data were obtained at room temperature in 0.01 M Tris- HC1 buffer of pH 7.0. RESULTS

Purification of a binding protein All the procedures were carried out at 4 °C unless otherwise stated. Step 1. Ultrasonic disruption. The cells (125 g, wet weight) were subjected to

157 ultrasonic disruption by suspending in 1.250 ml of 0.85 ~o NaC! using an Ultrasonic Disruptor, Model UR-200P (Tomy Seiko Co.) twice for 15 min. Cell debris was removed by centrifugation for 20 min at 17 000 x g, and streptomycin sulfate was added to the supernatant to the final concentration of 1 ~o. After 20 min at 0 °C, the precipitate was removed by centrifugation, and the supernatant was directly used for the second step. Step 2. (NH4)2S04 fractionation. To the supernatant (1400 ml) of the first step, (NH4)2SO4 (323 g) was added with stirring to a final concentration of 30 ~o saturation at 0 °C. After standing at 0 °C for 90 min, the precipitate (30 ~ precipitate) was removed by centrifugation, and (NH4)2SO4 (470 g) was added to the supernatant with stirring to a final concentration of 75 ~ saturation at 0 °C, and was kept standing overnight. Thereafter, the precipitate (75 ~ precipitate) was collected by centrifugation for 20 min at 17 000 × g. The 30~o precipitate (7.56 g protein) did not contain any significant amount of the protein which was adsorbed on an affinity column. The 75 ~ precipitate contained 5.35 g protein. Step 3. Sephadex G-75. The 75 ~o precipitate in the second step was dissolved in 110 ml of H 2 0 , and passed through a 5.0 × 87 cm column of Sephadex G-75 equilibrated with 0.05 M sodium phosphate buffer, pH 7.0, at a flow rate of 215 ml/h. The binding protein fractions (360 ml, 2.43 g protein) were concentrated by ultrafiltration to 45 ml using an Amicon TCF-10 with UM-2 membrane, and again passed through the same Sephadex G-75 column at a flow rate of 190 ml/h. The active fractions (335 ml, 1.67 g protein) were combined and concentrated to 7.5 ml using the UM-2 membrane. Step 4. The first affinity chromatography. The active concentrated fractions in the third step were passed through a cephalexin-CH-Sepharose 4B column [9] (1.6 × 35 cm) equilibrated with 0.05 M sodium phosphate buffer, pH 7.0, and the column was washed with the same buffer to the tube number 40 at a flow rate of 45 ml/h (7.5 ml/tube). Thereafter, the adsorbed proteins were eluted with a linear gradient of 0-1 M NaC1 (phosphate buffer without NaCl and with NaC1, 400 ml each) in the same buffer (7.5 ml/tube). The elution pattern is shown in Fig. la. The relevant fractions (No. 86 to No. 97, 90 ml) were concentrated to about 10 ml with concomitant dialysis against 0.05 M sodium phosphate buffer, pH 7.0, and were subjected to the second affinity chromatography. Step 5. The second qffinity chromatography. The active fractions in the fourth step were passed through the same affinity column equilibrated with the same buffer, and washed with the same buffer to the tube number 5 (18 ml/tube). Then, a linear gradient of 0-1 M NaC1 (phosphate buffer without NaC1 and with NaCl, 400 ml each) in the same buffer was applied, and each 7.5 ml fraction was collected. The elution pattern is shown in Fig. lb. The relevant fractions (No. 49 to No. 55) were concentrated to 5 ml with concomitant dialysis against 0.05 M sodium phosphate buffer, pH 7.0, using Amicon 8 MC with UM-10 membrane. The final yield was about 11.0 mg protein.

Physicochemical properties The purity of the binding protein purified in step 5 was checked by gel electrophoresis. In analytical polyacrylamide disc gel electrophoresis, the binding protein migrated as a single band (Fig. 2E) in a Tris/glycine buffer (pH 8.3) - 7.5~o poly-

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Fig. 1. Affinity chromatography of fl-lactamase and binding protein of C. freundii GN346. The active fraction which was separated by Sephadex G-75 column chromatography of cell protein collected from 31 1 culture was passed through a cephalexin-CH-Sepharose-4B column [9] equilibrated with 0.05 M sodium phosphate buffer, p H 7.0, and washed with the same buffer. Thereafter, the adsorbed proteins were eluted with a linear gradient of 0-1 M NaC1 in the same buffer. Fig. l a shows the elution pattern of the protein fractions. The volume of each fraction was 7.5 ml. Fraction A contains fl-lactamase. Fig. l b shows the elution pattern of rechromatography of fractions No. 86 to No. 97 obtained by previous chromatography. The volume of each fraction from No. 1 to No. 5 was 18 ml, and that of each fraction thereafter was 7.5 ml. fl-Lactamase activity was determined iodometrically at pH 7.0 using a slight modification [9, 10] of the method of Perret [11].

159 A

B

C

D

E

F

b

Fig. 2. Polyacrylamide disc gel electrophoresis of the binding proteins. Disc electrophoresis in the absence (A, B, C, D, and E) of sodium dodecyl sulfate was performed in a Tris/glycine buffer (pH 8.3)7.5 ~ polyacrylamide system and that in the presence (F) of sodium dodecyl sulfate were performed by the method of Weber and Osborn [7]. A, (NH4)2SO475 ~ precipitate; B, the active fraction from the first Sephadex G-75 column; C, the active fraction from the second Sephadex G-75 column; D: the fraction C in Fig. la; and E and F, the purified fraction in step 5.

acrylamide system. Disc electrophoresis in a sodium phosphate buffer (pH 7.0)-sodium dodecyl sulfate system [7] also resulted in the appearance of a single band (Fig. 2F). The molecular weight calculated from the mobilities in disc electrophoresis containing sodium dodecyl sulfate was 53 000. Fig. 2 shows the electrophoresis patterns of the proteins at the various purification steps. It is clear that the (NH4)2SO4 precipitate and the fractions from the Sephadex column contained the binding protein described above. Fig. 2D is a disc electrophoretic pattern of another cephalosporin-binding protein fraction, and shows at least 5 distinct protein bands.

fl-Lactamase activity The fl-lactamase activity of the purified binding protein in step 5 was determined against benzylpenicillin, ampicillin, cloxacillin, methicillin, cephalexin, cephaloridine, cephalothin, cephaloglycin, cephazoline, and cephalosporin C as substrates. Penicillins were added to the reaction mixture at a concentration of 6 m M and cephalosporins at 12 mM. In all cases, consumption of iodine was not observed at p H 5.8, 7.0 and 8.0. It is known [5] that the presence of some substrates, expecially cloxacillin and methicillin, reduces the hydrolysis rate of their own and other substrates by some fl-lactamases. Therefore, I tried to examine the fl-lactamase ac-

160 tivity of the binding protein in a substrate concentration of 100 #M without diluting the concentration of the binding protein by the spectrophotometric method. The decrease of the optical absorption was measured at 240 nm in the case of penicillins and at 260 nm in the case of cephalosporins. In all the cases, no indication of decrease of the optical absorption was observed in these wavelengths for over 10 min.

Binding of [14C]benzylpenicillin The binding of [~4C]benzylpenicillin to the binding protein was tested by passing the mixture of 0.44 mg of the binding protein and 1 mg of potassium salt of [a4C]benzylpenicillin (0.5/zCi) through a Sephadex G-25 column (0.9 × 51 cm) equilibrated with 0.01 M phosphate buffer of pH 7.0 at 0 °C. Portions of 1.2 ml fractions were collected and the optical absorption at 253.7 nm (Uvicord, LKB Co.) and the radioactivity were measured. The protein fractions were eluted in tube numbers 12 to 16 but no radioactivity was detected in these fractions. The radioactivity appeared only in benzylpenicillin-containing fractions (No. 25 to No. 31).

Amino acid composition Table I shows the amino acid composition of the binding protein. This indicates that the contents of aspartic and glutamic acids were much more than those of the basic amino acids. Methionine could not be detected. As for cysteine, some cysteic acid was detected, although the exact amount was not determined.

Fluorescence spectrophotometry Interaction of penicillins and cephalosporins with the binding protein was studied by fluorescence spectrophotometry. When benzylpenicillin, ampicillin or cloxacillin was added gradually to the solution of the binding protein in Tris. HCI buffer, TABLEI A M I N O ACID COMPOSITION OF A CEPHALOSPORIN-BINDING PROTEIN F R O M CITROBACTER F R E U N D H GN346 Amino acid

pmol/total

Number of residues per tool

24 h hydrolysate

41 h hydrolysate

Lys His Arg Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe

0.19 0.03 0.08 0.34 0.13 0.08 0.25 0.15 0.18 0.22 0.19 0.00 0.12 0.23 0.06 0.07

0.19 0.03 0.08 0.34 0.14 0.08 0.26 0.16 0.19 0.24 0.20 0.00 0.13 0.24 0.06 0.07

39 6 16 70 29 16 53 33 39 47 41 0 27 49 12 14

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Fig. 3. Scatchard plots [15] of the fluorescence data of the purified fraction in step 5. An excitation slit of 4 n m and an emission slit of 10 nm were used for all experiments. Emission spectra were obtained by an excitation wavelength of 290 nm, and monitoring was done at the peak near 335 nm. Data were obtained by a gradual addition of 15 m M or 30 m M of cephalosporins in 0.01 M Tris-HCI buffer, pH 7.0, at room temperature. Lines were calculated by the least-squares method. Fig. 3a shows A, cephalexin; B, cephaloridine; and C, cephalothin; and Fig. 3b shows D, cephaloglycin; E, cephazoline; and F, cephalosporin C. The right scale is for cephazoline, and the left side scale is for the other cephalosporins.

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162 none or only a slight quenching of the fluorescence was observed, or quenching of the fluorescence was variable. When binding data were plotted as (Fo/F -- 1)/[Q] vs [Q], where F0 and F are initial fluorescence intensity of the binding protein in the absence of penicillins and fluorescence intensity at any point in the titration, respectively, and [Q] is a concentration of penicillins, no straight line or a leaning towards a negative slope was observed. This means that, according to equation (1), no association occurred between penicillins and the binding protein [14].

Fo/F- 1 [Q]

- (Kq + K.) + Kq.K a. [Q]

(1)

where Kq and Ka are quenching and association constants, respectively. In contrast, addition of cephalosporins to the binding protein solution resulted in a gradual decrease of the fluorescence intensity, and finally almost no fluorescence was observed near 335 nm when the binding protein was saturated with cephalosporins. Plots of ( F o / F - 1)/[Q] vs [Q] gave straight lines with positive slopes in these cases. When titration data are treated by plotting as ~/[Q] vs ~, Scatchard plot [15] (Fig. 3), the number of binding sites, n, and Ka can be calculated (Eqn. 2).

[Q]

--

K..n

--

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where ~ is the average molar ratio of the bound ligand to the binding protein. Association constants and number of binding sites, calculated from these data by the leastTABLE II ASSOCIATION CONSTANTS AND NUMBERS OF BINDING SITES FOR VARIOUS CEPHALOSPORINS The values were calculated by the least-squares method from the data in Fig. 3. Cephalosporins

Association constants Number of binding sites (M -1)

Cephalexin Cephaloridine Cephalothin Cephaloglycin Cephazoline Cephalosporin C

0.83.103 3.1 • 10a 1.4 •103 1.6 • l03 12.0 • 103 1.3 •103

1.2 1.3 1.3 1.2 1.2 1.2

squares method, are shown in Table II. One binding site was shown for all the cephalosporins tested, and association constants were in the range of 0.8-12.103 M - L Cephazoline shows the strongest interaction, and cephalexin shows the weakest interaction with the binding protein. DISCUSSION A fl-lactamase from a strain of Citrobacterfreundii was reported [6] to show a broad and different optimum pH when penicillin and cephalosporin were used as

163 substrates. The broad optimum pH vs activity profile is not uncommon [16] in fllactamases. It suggests that the manner of the interaction of fl-lactamase and the other penicillin- and/or cephalosporin-binding proteins in this strain with penicillin would be different from that with cephalosporin. That is, the binding sites in these protein molecules change differently when these antibiotics come close to the binding site(s), or the binding sites for penicillin and cephalosporin are located in different places in these protein molecules. Keeping these ideas in mind, it was attempted to isolate penicillin- and/or cephalosporin-interacting proteins from this strain. This strain produces a fl-lactamase and in addition at least six distinct cephalexin-interacting proteins (Fig. 2), which can be easily obtained in soluble forms by ultrasonic disruption of the cells. From the supernatant of the sonicate, one of the cephalosporin-binding proteins could be obtained by affinity chromatography as a single band in analytical (Fig. 2E) as well as sodium dodecyl sulfate-containing disc electrophoresis (Fig. 2F). The molecular weight (53 000) did not appreciably differ from those of DD-carboxypeptidase of Bacillus subtilis [17] and of Streptomyces R39 (ref. 18). However, this cephalosporin-binding protein did not show any DD-carboxypeptidase activity. When the fl-lactamase activity was examined against various penicillins and cephalosporins at concentrations of 12 mM, 6 mM and 100#M, no indication of hydrolysis of these substrates was detected. This is not an unexpected result, because many penicillin-binding proteins without fl-lactamase activity have been isolated from a variety of bacteria [4, 19]. However, although it has not yet been examined, it is possible that such proteins have some other enzymatic or biological activity in bacterial cells. When the binding protein was titrated with several penicillins, almost no quenching was observed. In addition, plots of (Fo/F -- 1)/[Q] vs [Q] showed a leaning towards a negative slope. This indicates that these penicillins neither cause any change in charge distribution near tryptophan residues nor a conformational change. It also indicates that penicillin does not block tryptophan residues in the binding protein. On the contrary, addition of cephalosporins to the binding protein solutions resulted in a gradual decrease of the fluorescence intensity. Plots of (Fo/F-- l)/[Q] vs [Q] gave a straight line with a positive slope, and graphic calculation of the data by a Scatchard Plot [15] (Fig. 3) indicated one binding site with Ka values of 0.8 -12. l03 M -1 (Table II). It means that cephalosporins, in contrast to penicillins, cause a charge distribution change or a conformational change in the binding protein. It is reasonable to imagine that if penicillins bind to the same binding site of the binding protein, they should cause the same manner of quenching of tryptophan fluorescence. Even if penicillins and cephalosporins have different structures, it is unlikely that energy of the excited tryptophan residues is transferred to cephalosporins but not to penicillins. Thus, based on the data described above, it can be concluded that cephalosporins bind to the binding protein, but penicillins do not. The conclusion that penicillins do not bind to this binding protein was confirmed by gel filtration through Sephadex G-25. The binding protein obtained from a strain of C. freundii does discriminate between cephalosporins and penicillins. This protein is useful for the investigation of evolutionary and functional relationships of fl-lactamases to other penicillin- and cephalosporin-interacting proteins. It is tempting to speculate that the binding site(s) for penicillins and cephalosporins in some fl-lactamases and penicillin-

164 a n d / o r c e p h a l o s p o r i n - b i n d i n g proteins are evolved or modified independently. In this connection, it is interesting that the fl-lactamase from this strain was not adsorbed on an affinity c o l u m n u n d e r conditions where other fl-lactamases were easily adsorbed. ACKNOWLEDGMENTS The a u t h o r would like to express his great thanks to Dr. H a m a o Umezawa, Head of the D e p a r t m e n t , for his hearty and courteous e n c o u r a g e m e n t a n d discussion t h r o u g h o u t the present work, a n d to Professor S u s u m u Mitsuhashi, G u n m a University, for his kind supply of a strain of Citrobacter freundii. REFERENCES 1 Ghuysen, J. M., Leyh-Bouille, M., Frere, J. M., Dusart, J., Johnson, K., Nakel, M. and Coyette, J. (1971) in "Molecular mechanisms of antibiotic action on protein biosynthesis and membranes" (Munoz, E., Garcia-Ferrandiz, F. and Vazquez, D., ed.), pp. 406~26, Elsevier Scientific Publishing Co., Amsterdam 2 Pollock, M. R. (1971) Proc. Roy. Soc. London B, 179, 385-401 3 Saz, A. K. (1970) J. Cell Physiol. 76, 397-404 4 Strominger, J. L., Willoughby, E., Kamiryo, T,, Blumberg, P. M. and Yocum, R. R. (1974) Ann. New York Acad. Sci. 235, 210-224 5 Citri, N. (1971) in "The Enzymes" (Boyer, P. D., ed.), Vol. IV, pp. 23-46, Academic Press, New York 6 Sawai, T., Mitsuhashi, S. and Yamagishi, S. (1970) in "Progress in Antimicrobial and Anticancer Chemotherapy (Proc. 6th Int. Congr. Chemotherapy)", Vol. I, pp. 410-415, University of Tokyo Press, Tokyo 7 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 9 Ogawara, H. and Umezawa, H. (1975) Biochim. Biophys. Acta 391, 435-447 10 Ogawara, H. (1975) Antimicrob. Agents and Chemotherapy, in the press 11 Perret, C. J. (1954) Nature 174, 1012-1013 12 O'Callaghan, C. H., Muggleton, P. W. and Ross, G. W. (1969) Antimicrob. Agents and Chemotherapy 1968, pp. 57-63 13 Samuni, A. (1975) Anal. Biochem. 63, 17-26 14 Chen, R. F. and Cohen, P. F. (1966) Arch. Biochem. Biophys. 114, 514-522 15 Scatchard, G., Scheinberg, I. H. and Armstrong, S. H. (1950) J. Am. Chem. Soc. 72, 535-540 16 Furth, A. J. (1975) Biochim. Biophys. Acta 377, 431-443 17 Blumberg, P. M. and Strominger, J. L. (1972) J. Biol. Chem. 247, 8107-8113 18 Frere, J. M., Ghuysen, J. M., Perkins, H. R. and Nieto, M. (1973) Biochem. J. 135,463-468 19 Blumberg, P. M. and Strominger, J. L. (1974) Bacteriol. Rev. 38. 291-335

A specific cephalosporin-binding protein of Citrobacter freundii.

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium...
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