©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biosynthesis of the Macrolide Oleandomycin by Streptomyces antibioticus
PURIFICATION AND KINETIC CHARACTERIZATION OF AN OLEANDOMYCIN GLUCOSYLTRANSFERASE (*)

(Received for publication, March 29, 1995; and in revised form, May 1, 1995)

Luis M. Quirs (§) Jos A. Salas (¶)

From the Departamento de Biologa Funcional e Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular inactivation and extracellular reactivation of the antibiotic. Inactivation takes place by transfer of a glucose molecule from a donor (UDP-glucose) to OM, a process catalyzed by an intracellular glucosyltransferase. Glucosyltransferase activity is detectable in cell-free extracts concurrent with biosynthesis of OM. The enzyme has been purified 1,097-fold as a monomer, with a molecular mass of 57.1 kDa by a four-step procedure using three chromatographic columns. The reaction operates via a compulsory-order mechanism. This has been shown by steady-state kinetic studies using either OM or an alternative substrate (rosaramycin) and dead-end inhibitors, and isotopic exchange reactions at equilibrium. OM binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated oleandomycin (GS-OM).


INTRODUCTION

The biosynthesis of a potentially lethal antibiotic necessitates the existence of a self-resistance mechanism in the producing organism. One of the most common (for review, see (1) ) involves modification of the antibiotic target site, for example, inhibitors of ribosomal function(2, 3, 4, 5, 6, 7) , RNA polymerase(8, 9, 10) , DNA gyrase (11) , elongation factor EF-Tu(12) , and fatty acyl synthase(13) . Activities capable of inactivating antibiotics in vitro have also been described. However, despite the structural diversity of the different antibiotic families, most of the inactivating activities so far described catalyze the N-acetylation of amino groups or O-phosphorylation of hydroxyl groups using acetyl-coenzyme A and ATP, respectively, as donor cofactors. Most of these activities have been reported in aminoglycoside producers (reviewed in (1) ). Inactivation of fosfomycin by a glutathione S-transferase in the producer strain has also been reported(14) . A possible role for the inactivating activities in the producer strains is their participation in the antibiotic biosynthetic pathway, being in fact biosynthetic enzymes. In the case of puromycin, biochemical evidence suggests a role for puromycin N-acetyltransferase in the biosynthesis of the antibiotic(15) .

We have previously reported the existence of a dual mechanism for inactivation and reactivation of OM in the oleandomycin (OM) (^1)producer, Streptomyces antibioticus(16) . This strain synthesizes an intracellular glucosyltransferase (GTF) enzyme capable of inactivating OM (and a few other macrolides) by glucosylation of the 2`-hydroxyl group of the desosamine present in the antibiotic (Fig. 1). This glucosylated oleandomycin (GS-OM) is the final intracellular product of the pathway. We have proposed that this inactive GS-OM could be secreted through participation of ABC (``ATP Binding Cassette'') transporters encoded by the oleB and oleC genes(18) . In agreement with this, recent experimental evidence has demonstrated that the OleB protein is capable of pumping out GS-OM(17) . To complete OM biosynthesis, S.antibioticus synthesizes and secretes a second enzyme that has recently been purified(19) . This reactivates the antibiotic by catalyzing release of glucose.


Figure 1: Chemical structure of OM and proposed site of action (indicated by the arrow) for the GTF.



Very little is known at present about the reaction mechanisms of enzymes involved in antibiotic biosynthesis and resistance. Here we report the purification and characterization of the OM GTF and kinetic studies to obtain a more detailed knowledge of the enzymatic mechanism of this GTF.


MATERIALS AND METHODS

Reagents

Oleandomycin, erythromycin, and UDP-glucuronic acid agarose were purchased from Sigma. Rosaramycin was purchased from Schering Corp. UDP-[6-^3H]glucose (specific activity 7.6 Ci/mmol, 1 mCi/ml) from Amersham International (United Kingdom). UDP-glucose, UTP, and dithiothreitol (DTT) were from Sigma. Acrylamide and bisacrylamide were from Bio-Rad. Acetonitrile (HPLC grade) and ammonium sulfate from Merck, and Q-Sepharose, Sephacryl S-200, and Superdex 75 from Pharmacia Biotech Inc. (Uppsala, Sweden). All other chemicals were obtained from commercial sources and were of analytical grade.

Purification Procedure

S. antibioticus ATCC 11891, an OM producer, was grown in 2-liter Erlenmeyer flasks containing 500 ml of TSB (tripticasein soy broth, Oxoid) liquid medium. The cultures were inoculated with 50 µl of a dense spore suspension and after 48 h at 30 °C on an orbital shaker incubator (200 rpm), the mycelia were collected by filtration through Whatman No. 1 filters. The mycelial paste was washed twice with buffer A (50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA, and 1 mM DTT). The mycelia was then disrupted by homogenization with glass beads (0.10 mm in diameter) in a Bead Beater (Bead Beater, Bartlesville, OK) for 10 15-s periods with 3-min intervals for cooling. Unbroken cells and debris were removed by centrifugation at 14,000 g for 30 min. Nucleic acids were precipitated with streptomycin sulfate (1% final concentration) and the supernatant fractionated by precipitation with ammonium sulfate at 50% saturation. After centrifugation (14,000 g, 30 min), the supernatant was dialyzed against buffer A and applied to a Q-Sepharose column (200 ml volume) at a flow rate of 3 ml/min. The column was eluted with a gradient of 0-1 M NaCl. Active fractions were concentrated by ammonium sulfate precipitation (95% saturation) and, after centrifugation, the precipitate was resuspended in 5 ml of buffer B (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, and 0.5 M ammonium sulfate) before dialysis against buffer B. The sample was then applied to a Sephacryl S-200 column (2.6 90 cm) at a flow rate of 0.3 ml/min. Active fractions were dialyzed against buffer C (25 mM Tris-HCl, pH 7.5, 1 mM DTT, and 20% glycerol) and then applied to a UDP-glucuronic acid-agarose column (10 ml) at a flow rate of 0.3 ml/min. Elution was with a 0-1 M NaCl gradient in buffer C.

Enzyme Assays

OM inactivation was monitored using a reaction mixture containing (for a 50-µl final volume): 1 µl of OM (3 µM final concentration), 1 µl of UDP-[^3H]Glc (0.26 µM final concentration), and 1 µl of UDP-Glc (22 µM final concentration), and a variable volume of each fraction or buffer. The mixture was incubated at 30 °C for 3 min and the pH subsequently raised by addition of 5 µl of 0.2 M NaOH. It was then extracted with 50 µl of chloroform, following centrifugation at 12,000 g for 5 min in an Eppendorf minifuge, 25 µl of the lower organic phase were evaporated to dryness and resuspended in 50 µl of methanol. Any radioactivity measured was indicative of the inactivating activity.

Biological Assay of OM

The biological activity of OM was tested by performing a bioassay against Micrococcus luteus ATCC 10240(20) .

Purification of GS-OM

After performing glucosylation reactions as described above, the pH of the reaction mixture was raised by adding 0.2 M NaOH (0.1, v/v). This was extracted with 1 volume of chloroform. After centrifugation, the organic phase was recovered, flash evaporated, and suspended in methanol:water (1:1 by volume), before application to a µBondapak C(18) column. GS-OM and OM were separated using an isocratic gradient composed of 30% acetonitrile and 70% 50 mM phosphate buffer, pH 6.8, at a 2 ml/min flow rate. Detection was followed by measuring absorbance at 200 nm. Fractions corresponding to GS-OM were collected, dried, and resuspended in chloroform so as to eliminate residual phosphate. The chlorofom was then removed by evaporation and the residue (containing the GS-OM) dissolved in methanol:water (1:1 by volume) and stored at -20 °C prior to use.

Polyacrylamide Gel Electrophoresis and Protein Analysis

Analysis of the samples during protein purification was performed using SDS-PAGE(21) . Protein estimation was carried out by measurement of absorbance at 280 nm and by a protein-dye binding assay (22) .

Steady State Kinetics

Initial velocity assays were carried out using the radiolabeled assay described above. The concentration of one substrate was varied while the concentration of the second substrate was held at a nonsaturating concentration. After stopping the reaction, the amount of GS-OM produced was estimated. The experimental data reported here were all carried out at least in duplicate. Data were analyzed by linear and nonlinear regression using the program Statistica for Windows (Statsoft, Inc.).

Measure of Isotopic Exchange Reactions Catalyzed by the GTF

The exchange reactions between UDP-[^3H]Glc and [^3H]GS-OM were assayed by measuring the formation of [^3H]GS-OM from UDP-[^3H]Glc and unlabeled OM. Assays were performed as described under ``Steady State Kinetics,'' except that the concentrations of unlabeled reactants and products were adjusted to the equilibrium conditions at the beginning of the experiment. The concentrations for the unchanged pair were 0.64 and 5 µM, and the concentrations of the changed pair were varied from 0.5 to 40 times the concentration of the other pair depending on the assay. After addition of enzyme, the reaction mixture was incubated at 30 °C for 15 min. A small concentration (10 nM) of UDP-[^3H]Glc was added subsequently, and samples taken at different times and used to calculate the rate of isotopic exchange.


RESULTS AND DISCUSSION

Time Course for the Synthesis of OM and the GTF

OM production began 24 h after inoculation of S. antibioticus in TSB medium (Fig. 2) and increased progressively during the following 24 h. In cell-free extracts GTF activity was also detected after 24 h of growth, which coincided with the beginning of OM biosynthesis. The GTF activity increased during the next 24 h following a similar pattern to that of OM biosynthesis, suggesting, therefore, that both events are coordinately regulated.


Figure 2: Time course for the biosynthesis of OM (bullet) and the OM GTF ([circo]) during growth of S.antibioticus. At different times of incubation of S.antibioticus in TSB medium, samples were removed and OM present in the supernatant determined by bioassay against M.luteus. The mycelia was collected and the GTF activity assayed using a cell-free extract (see ``Materials and Methods'').



Cellular Location of the GTF

To determine the physical location of the GTF, protoplasts were prepared by lysozyme digestion (23) and gently lysed by 1:10 dilution into a nonstabilizing buffer (50 mM Tris-HCl, pH 8.5, 1 mM DTT) containing either 150 mM or 1 M NaCl. The samples were ultracentrifuged (100,000 g) for 30 min and the GTF activity assayed in fractions along the tube. Activity was distributed uniformly, and was independent of the salt concentration used in protoplast lysis (data not shown), indicating the absence of any association between GTF and particulate fractions (i.e. membranes).

Purification and Characterization of the GTF

The GTF enzyme was purified by a four-step purification procedure using three chromatographic columns (Fig. 3). A summary of the purification procedure is shown in Table 1. The enzyme was eluted from the Q-Sepharose column at 0.33 M NaCl (Fig. 3A) and from the UDP-glucuronic acid-agarose column at 0.12 M NaCl (Fig. 3C). The enzyme was purified 1,097-fold with an 18% yield. It showed a molecular mass of approximately 57.1 kDa as deduced from SDS-PAGE gels (Fig. 4) and 56 kDa as calculated by gel filtration on a Superdex 75 column in the presence of 0.15 M NaCl at a flow rate of 0.1 ml/min (data not shown). These experiments suggests that the GTF is a monomer. The enzyme was moderately unstable when kept at low temperature, losing almost all activity after a few days at 4 °C, although the addition of 1 M ammonium sulfate or 20% glycerol was found to stabilize the enzyme. Activity was maximal between pH 8 and 8.5. Enzyme activity was shown to be unaffected by the addition of different mono or divalent cations, but was affected by the addition of low concentrations of several organic solvents such as methanol, ethanol, butanol, and acetone. This contrasts with the purified extracellular glycosidase which is active in the presence of as much as 20% acetone or ethanol(19) .


Figure 3: Chromatography of the different steps in the purification of the GTF. Elution profile from (A) the Q-Sepharose column, (B) the Sephacryl S-200 column, and (C) the UDP-glucuronic acid agarose column. bullet, GTF activity; bulletbulletbullet, salt gradient; --, absorbance at 280 nm.






Figure 4: Silver-stained SDS-PAGE analysis of the different steps in the purification of the GTF activity. Lane A, cell-free extract; lane B, ammonium sulfate precipitation; lane C, active fractions eluted from the Q-Sepharose column; lane D, active fractions eluted from the Sephacryl S-200 column; lane E, active fractions eluted from the UDP-glucuronic acid agarose.



Steady State Initial Velocity Studies

For most of the reaction mechanisms involving a two-substrate enzyme, the initial rate is given, according to the nomenclature of Cleland(24) , by the following equation:

where A and B signify the first and second substrates, respectively, that bind to the enzyme, V is V(max), K and K are the K values for A and B, respectively, and Kis the dissociation constant for the reaction of A with the free enzyme. A plot of 1/vversus 1/[substrate] showed a set of converging lines, when the substrate was either UDP-Glc (Fig. 5A) or [OM] (Fig. 5B) at fixed concentrations of the other substrate. The following parameters were obtained by performing nonlinear regression analysis of the initial velocity data: V = 363.8 ± 27 nmolbulletminbulletmg protein; K = 2.9 ± 0.19 µM, K = 21.57 ± 2.3 µM, K = 0.44 ± 0.06 µM. A set of intersecting lines in the analysis of the initial velocity steady state kinetic data is indicative of a sequential mechanism in which a ternary complex is formed prior to product release.


Figure 5: Double-reciprocal plots of the initial velocities with variable substrate concentrations. A, variable UDP-Glc at fixed OM concentrations. bullet, 7 µM; , 3.5 µM; ▪, 2.3 µM; , 1.75 µM; ▴, 1 µM. B, variable OM at fixed UDP-Glc concentrations. bullet, 32 µM; , 16 µM; ▪, 8 µM; , 5.2 µM; ▴, 3.1 µM; , 2.2 µM.



Several assays were performed in which one of the substrates (OM) was replaced by rosaramycin. Rosaramycin is a macrolide that is related structurally to OM and has been described as an alternative substrate for the GTF(16) . When rosaramycin was used instead of OM, convergence between lines became more apparent (data not shown), due to the change in the kinetic parameters. The values of the new parameters obtained by nonlinear regression were: V = 232 ± 31 nmolbulletminbulletmg protein, K = 13.22 ± 2.1 µM, K = 42.09 ± 3.8 µM, K = 11.14 ± 1.6 µM. An increase in the value of K close to that of K makes the convergence between the lines much more evident and places the intersection point just below the horizontal axis.

However, a graphical analysis of the results of both experiments expressed in reciprocal form in terms of Ø's according to the equation of Daziel(25) :

showed that all the values of the Ø's changed when rosaramycin was used as alternative substrate. This indicates OM binds the enzyme in the first place (substrate A), based on the fact that the use of an alternative substrate for B would maintain some of the coefficients constant depending on the mechanism of the reaction. It can also be concluded that the reaction occurs by a compulsory-order mechanism, due to the fact that in the random order equilibrium pathway Ø(A) might be unaltered when using an alternative substrate for A.

Study of Substrate Binding Order Using Dead-end Inhibitors

Dead-end inhibitors were used to obtain information about the mechanism of the reaction and the substrate binding order. The concentration of one of the substrates was fixed at a subsaturating level, and the concentration of the other substrate varied; experiments were performed at different concentrations of the dead-end inhibitor, including 0.

The antibiotic erythromycin is structurally very similar to OM, but it is not a substrate for the GTF(16) . When erythromycin was added to the reaction using OM as the varied substrate, a set of lines of different slopes was observed, while the intersection point remained constant, thus indicating a competitive inhibition (Fig. 6A). If the substrate varied was UDP-Glc, the lines changed, both in their slope and intersection point (Fig. 6B), implying noncompetitive inhibition.


Figure 6: Patterns of dead-end inhibition of the GTF by erythromycin (panels A and B) or by UTP (panels C and D) as a function of the OM (panels A and C) or UDP-Glc (panels B and D) concentrations. The concentrations of UDP-Glc and OM were kept constant at 8 µM (panels A and C) and 2 µM (panels B and D), respectively. The concentrations of erythromycin as inhibitor (panels A and B) were: bullet, 0 µM; , 2.5 µM; ▪, 5 µM; , 10 µM. The concentrations of UTP as inhibitor (panels C and D) were: bullet, 0 µM; , 25 µM; ▪, 67 µM; , 200 µM.



UTP was also found to act as a dead-end inhibitor of the reaction. When reactions were carried out in the presence of UTP using UDP-Glc as the varied substrate, the lines changed their slopes but the intersection point remained constant, suggesting competitive inhibition (Fig. 6D). If the varied substrate was OM, the lines were parallel, with unchanged slope but with changed point of intersection, indicating uncompetitive inhibition (Fig. 6C).

Data from dead-end inhibitors are summarized in Table 2. The patterns of inhibition observed showed noncompetitive inhibition for erythromycin when UDP-Glc is varied and uncompetitive inhibition for UTP when OM is varied, and this is compatible only with a compulsory-order mechanism. It does not, however, differentiate between formation of a ternary complex or if its concentration is negligible (Theorell-Chance mechanism). On the other hand, these results allow confirmation of the order of substrate addition, OM being first and UDP-Glc second.



Equilibrium Constant

The equilibrium constant for the GTF reaction might be expected to be independent of pH as no net production or uptake of protons occurs during the course of the transglucosylation reaction. The time taken to reach equilibrium was first established by measurement of the change in the concentration of GS-OM with time after addition of the enzyme to solutions similar to those described below. Reaction was always completed in less than 1 h. When sampled after several hours, the concentration of GS-OM began to decrease, due to its instability at 30 °C. K was measured by fixing the ratio of [OM]/[GS-OM] at 1 and varying the ratio of [UDP]/[UDP-Glc] from 10 to 80. A plot of the change in GS-OM concentration versus [UDP]/[UDP-Glc] is shown in Fig. 7. The value at which the line intercepts with the ordinate axis for an abscissa value of 0 is equal to the equilibrium constant K = 60.81.


Figure 7: Plot of Delta[GS-OM] versus [UDP]/[UDP-Glc] for determining the equilibrium constant of the GTF reaction. A linear regression of the data gave a [UDP]/[UDP-Glc] value of 60.81 at a [GS-OM] of 0.



Reactions of Isotopic Exchange at Equilibrium

To obtain information about the order of products release and more details about the reaction mechanism, we performed isotopic exchange experiments under equilibrium conditions. The concentration of a substrate-product pair was varied so as not to disturb the equilibrium; its effect on the rate of isotopic exchange between UDP-[^3H]Glc and [^3H]GS-OM was then determined. The progress curve for the exchange, monitored by sampling the reaction mixture at different time intervals, showed in all the cases a first-order rate law; these curves were used to calculate the rates of exchange. The exchange rate was also directly proportional to enzyme concentration, as would be expected in the absence of subunit dissociation and association effects.

When the effect of increased concentrations of the pair OM/GS-OM on the rates of exchange between UDP-[^3H]Glc and [^3H]GS-OM was measured, a hyperbolic plot was obtained (Fig. 8A). A similar experiment varying the pair OM/UDP produced a plot showing an initial increase followed by a decrease due to inhibition of the isotopic exchange at high concentrations of the pair OM/UDP (Fig. 8A). These results are consistent only if the order of product release is the one shown in Fig. S1, where UDP leaves first followed by GS-OM. Raising the concentration of the pair OM/UDP makes the enzyme-GS-OM and enzyme species scarce, increasing other forms, and the exchange rates are lowered. If the concentration of the OM/GS-OM pair was raised, only the free enzyme becomes scarce and the rates of exchange approach a maximum asymptotically.


Figure 8: Influence of increasing concentrations of different substrate/product pairs on the rate of exchange between UDP-Glc and GS-Om. A: bullet, varying the pair OM/UDP; , varying the pair OM/GS-OM. B: bullet, varying the pair UDP-Glc/UDP; , varying the pair UDP-Glc/GS-OM.




Figure S1: Scheme 1.



The differentiation between a compulsory-order system with the formation of a ternary complex and a Theorell-Chance mechanism is possible by performing experiments where the influence on the rate of isotopic exchange is measured when the concentration of the pairs composed by the second substrate and one of the different products is increased. Increasing the concentration of the UDP-Glc/GS-OM pair a hyperbolic plot was produced (Fig. 8B), while the increase of the UDP-Glc/UDP pair produced a plot which initially increased but later showed a depression at higher concentrations (Fig. 8B). These results confirm the order of the product release from the enzyme, since raising the concentrations of the UDP-Glc/UDP pair increases the concentrations of the central enzymic forms, and depresses the rate of isotopic exchange. This is compatible only with an ordered mechanism in which a ternary complex is formed. In a Theorell-Chance mechanism, the increase in the UDP-Glc/UDP pair would produce a hyperbolic plot since in this mechanism the process EA + B EQ &lrhar2; + P is a simple bimolecular process.

In summary, the mechanism of the GTF reaction can be represented as in Fig. S1. The substrates bind in a compulsory order to the enzyme, first the OM and then the UDP-Glc to form a ternary complex in which the exchange reaction takes place. Product release also occurs in a compulsory order, UDP first followed subsequently by GS-OM.


FOOTNOTES

*
This work was supported in part by a grant of the Plan Nacional de Biotecnologa (BIO91-0758). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a grant from the FICYT, Asturias (Spain), and a contract from the Spanish Ministery of Education and Science.

To whom correspondence should be addressed. Tel.: 34-85-103652; Fax: 34-85-103148; jasf{at}dwarf1.quimica.uniovi.es.

^1
The abbreviations used are: OM, oleandomycin; GS-OM, glucosylated oleandomycin; DTT, dithiothreitol; GTF, glucosyltransferase; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Schering Corp. for the kind gift of rosaramycin.


REFERENCES

  1. Cundliffe, E. (1989)Annu. Rev. Microbiol.43,207-233 [CrossRef][Medline] [Order article via Infotrieve]
  2. Yamamoto, H., Hotta, K., Okami, Y., and Umezawa, H.(1981)Biochem. Biophys. Res. Commun.100,1396-1401 [Medline] [Order article via Infotrieve]
  3. Skinner, R. H., and Cundliffe, E.(1982)J. Gen. Microbiol.128,2411-2416
  4. Zalacain, M., and Cundliffe, E.(1989)J. Bacteriol.171,4254-4260 [Medline] [Order article via Infotrieve]
  5. Zalacain, M., and Cundliffe, E.(1990)Eur. J. Biochem.189,67-72 [Abstract]
  6. Zalacain, M., and Cundliffe, E.(1991)Gene (Amst.) 97,137-142 [CrossRef][Medline] [Order article via Infotrieve]
  7. Piendl, W., and Bock, A. (1982)Antimicrob. Agents Chemother.22,231-236 [Medline] [Order article via Infotrieve]
  8. Watanabe, S., and Tanaka, K.(1976)Biochem. Biophys. Res. Commun.72,522-529 [Medline] [Order article via Infotrieve]
  9. Blanco, M. G., Hardisson, C., and Salas, J. A.(1984)J. Gen. Microbiol. 130,2883-2891 [Medline] [Order article via Infotrieve]
  10. Roza, J., Blanco, G., Hardisson, C., and Salas, J. A.(1986)J. Antibiot. 39,609-612 [Medline] [Order article via Infotrieve]
  11. Thiara, A. S., and Cundliffe, E.(1988)EMBO J.7,2255-2259 [Abstract]
  12. Glckner, C., and Wolf, H.(1984)FEMS Microbiol. Lett. 25,121-124
  13. Kawaguchi, A., Tomoda, H., Okuda, S., Awaya, S., Omura, S.(1979)Arch. Biochem. Biophys.197,30-35 [Medline] [Order article via Infotrieve]
  14. Arca, P., Rico, M., Braa, A. F., Villar, C. J., Hardisson, C., and Surez, J. E.(1988)Antimicrob. Agents Chemother.32,1552-1556 [Medline] [Order article via Infotrieve]
  15. Vara, J., Prez-Gonzlez, J. A., and Jimnez, A.(1985)Biochemistry 24,8074-8081 [Medline] [Order article via Infotrieve]
  16. Vilches, C., Hernndez, C., Mndez, C., and Salas, J. A.(1992)J. Bacteriol.174,161-165 [Abstract]
  17. Olano, C., Rodriguez, A. M., Mndez, C., and Salas, J. A.(1995) Mol. Microbiol.16,333-343 [Medline] [Order article via Infotrieve]
  18. Rodriguez, A. M., Olano, C., Vilches, C., Mndez, C., and Salas, J. A. (1992)Mol. Microbiol.8,571-582
  19. Quirs, L. M., Hernndez, C., and Salas, J. A. (1994)Eur. J. Biochem.222,129-135 [Abstract]
  20. Vilches, C., Mndez, C., Hardisson, C., and Salas, J. A. (1990)J. Gen. Microbiol.136,1447-1454 [Medline] [Order article via Infotrieve]
  21. Laemmli, U. K. (1971)Nature227,680-685
  22. Bradford, M. M. (1976)Anal. Biochem.72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  23. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M., and Schrempf, H. (1985) Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Norwich, England
  24. Cleland, W. W. (1963)Biochim. Biophys. Acta67,104-137 [CrossRef]
  25. Daziel, K.(1957) Acta Chem. Scand.11,1706-1712

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.