©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hemolytic, but Not Cell-invasive Activity, of Adenylate Cyclase Toxin Is Selectively Affected by Differential Fatty-acylation in Escherichia coli(*)

(Received for publication, June 15, 1995)

Murray Hackett (1) Carthene B. Walker (1) Lin Guo (2) Mary C. Gray (2) Sheila Van Cuyk (2) Agnes Ullmann (4) Jeffrey Shabanowitz (1) Donald F. Hunt (1) (3) Erik L. Hewlett (2)(§) Peter Sebo (4)(¶)

From the  (1)Departments of Chemistry, (2)Medicine and Pharmacology, and (3)Pathology, University of Virginia, Charlottesville, Virginia 22901 and the (4)Unite de Biochimie des Regulations Cellulaires, Institut Pasteur, F-75724 Paris Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.


INTRODUCTION

Adenylate cyclase (AC) (^1)toxin is a bifunctional 177-kDa bacterial protein that contains a calmodulin-activated adenylate cyclase catalytic domain and a pore-forming domain. It exhibits a cell-invasive activity by delivering its catalytic domain to the interior of target cells to elicit supraphysiologic cAMP accumulation(1, 2, 3, 4) . The pore-forming capacity of the molecule is responsible for hemolytic activity against sheep erythrocytes and is entirely independent of the presence and activities of the catalytic domain(5, 6, 7, 8, 9) .

The AC toxin gene (cyaA) has been cloned and sequenced by Glaser et al.(10, 11) and the AC toxin locus was noted to have homologies with the Escherichia coli hemolysin operon and that of other RTX toxins. These toxins are characterized by a set of glycine- and aspartate-rich nonapeptide repeats(12, 13) . The formation of biologically active AC toxin requires an accessory protein expressed from an upstream gene, cyaC(14) . That protein, CyaC, appears to be involved in posttranslational activation of AC toxin. Recently, tandem mass spectrometry was used to determine that the modification on native AC toxin from Bordetella pertussis (Bp-CyaA) consists of palmitoylation on the -amino group of Lys(15) . In vitro random chemical acylation has also been shown to confer limited cell-invasive and hemolytic activities on CyaA(16) .

It has been observed previously that recombinant AC toxin expressed in E. coli exhibits cell-invasive activity identical to the native toxin, but hemolytic activity which is severalfold reduced(17) . In order to determine whether the nature of the acylation could be the basis of this functional difference, wild type AC toxin from B. pertussis and recombinant AC toxin from E. coli were analyzed by mass spectrometry. We report here that despite the presence of the same requisite CyaC, the pattern and chemical nature of the acylation are different when AC toxin (CyaA) is expressed in these two organisms.


MATERIALS AND METHODS

Purification of Toxins

The four proteins evaluated in this study are designated as follows: Bp-CyaA, the native CyaC-activated AC toxin from B. pertussis; Bp-proCyaA, the non-activated AC protoxin from strain BPDE386 lacking functional CyaC; r-CyaA, the recombinant CyaC-activated AC toxin produced in E. coli; and r-proCyaA, the recombinant non-activated AC protoxin produced in E. coli lacking CyaC. The E. coli strain XL1-Blue (Stratagene) was used for expression of r-CyaA from the pCACT3 and for expression of r-proCyaA from pACT7(17, 18) . Strains BP338 and BPDE386 were used for production of Bp-CyaA and Bp-proCyaA, respectively(14) .

The fermentor cultures of E. coli strains were supplied by the Service of Fermentations of Institut Pasteur (Paris). The r-CyaA and r-proCyaA were extracted from E. coli cell debris after French press disruption, as described previously(17) . Bp-CyaA was extracted from washed B. pertussis cells with 4 M urea as described previously(5) . The different CyaAs were purified close to homogeneity by a combination of ion-exchange chromatography (19) and affinity chromatography(17) . In the final step CyaAs were eluted from calmodulin-agarose columns with 8 M urea, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and frozen at -70 °C.

Assays of Adenylate Cyclase Enzymatic, Invasive, and Hemolytic Activities

Adenylate cyclase enzymatic activity was measured as described previously(20) . One unit of activity corresponds to 1 µmol of cAMP formed in 1 min at 30 °C, pH 8. Hemolytic activity and the cell-invasive activity, which has been shown to be a good measure of intoxicating activity of CyaAs, were both determined using sheep erythrocytes (5 10^8/ml) essentially as described(5) .

Enzymatic Digestions

Recombinant and B. pertussis expressed AC toxin preparations were digested with sequencing grade modified trypsin (Promega) or sequencing grade Asp-N (Boehringer Mannheim) at an enzyme:substrate ratio of approximately 1:50 (w/w) in Tris adjusted to pH 8.5 for trypsin and 8.0 for Asp-N, 0.2 ml total volume. All digests were carried out for 8 h at 37 °C, adjusted in volume to 0.5 ml with 10% acetonitrile, acidified with trifluoroacetic acid, and frozen at -40 °C until HPLC fractionation.

Microbore HPLC and Screening by MALDI-TOF Mass Spectrometry

All r-CyaA digests and Asp-N digests of Bp-CyaA were separated with a Polymer Laboratories PLRP-S 8 µm 300 Å 2.1 250 mm column using a reversed-phase binary gradient(21) . HPLC conditions for the fractionation of the tryptic fragments derived from Bp-CyaA have also been described previously(15) . Each fraction was screened for the molecular mass of the fragments using MALDI-TOF mass spectrometry with an instrument built in-house(31) , as described previously(15, 21) . Calculations for predicting peptide mass values were performed using Mac ProMass. (^2)

Combined Microcapillary HPLC Electrospray Ionization Mass Spectrometry

HPLC Fractions from the tryptic digests which were shown by MALDI-TOF MS to contain peptides which might possess a posttranslational modification were analyzed further for amino acid sequence (22, 23) by tandem quadrupole mass spectrometry using an upgraded TSQ70 instrument with a Finnigan electrospray source, as described(21) . The molecular masses of the Asp-N fragments of interest were confirmed by electrospray. To obtain semi-quantitative estimates of the relative amounts of palmitoylation versus myristoylation at Lys, and palmitoylation versus unmodified peptide at Lys, the following procedures were used. For the two tryptic acyl peptides containing Lys, the relevant HPLC fractions were adjusted to 100 µl, a 10-µl aliquot removed from each fraction, and the aliquots pooled into two 1.5-ml microcentrifuge tubes, one containing the total myristoylated peptide, the other the total palmitoylated peptide. Each tube was adjusted in volume to 50 µl using a solution of 10% acetonitrile, 0.1% trifluoroacetic acid. Three replicate 1-µl injections were made using the microcapillary HPLC apparatus on-line with the TSQ70, as described previously(15, 21, 23) . For each determination of the myristoyl peptide, the centroid signals from ions with +3, +2, and +1 charge states were summed, m/z 532, 797, and 1593. Similarly for the palmitoyl peptide, m/z 541, 811, and 1621 were summed. The average of the three sums was calculated and the precision estimated by calculating a pooled RSD(24) . The same approach was used for the Lys-containing peptide, summing the five most abundant charge states for the unmodified Asp-N peptide at m/z 5120, +5 to +9. For the palmitoylated Asp-N fragment at m/z 5358, the charge states from +4 to +8 were summed.

Synthesis of Analytical Standards

Peptides were synthesized using automated Fmoc chemistry (25) with a Gilson 422 synthesizer and purified by analytical scale reversed-phase HPLC, according to a published procedure(21) . The modified peptides were selectively acylated while still attached to the Wang resin (Novabiochem) at the -amino group of the desired lysine residue by the use of alpha-(1-4,4-dimethyl-2,6-dioxocyclohexylidine)-ethyl--Fmoc-lysine (Novabiochem)(32) . The purified synthetic peptides were used to confirm molecular masses, CAD-derived sequence information, and HPLC retention times.


RESULTS AND DISCUSSION

CyaC-activated recombinant AC toxin (r-CyaA) was produced in E. coli(17, 18) and compared with Bp-CyaA extracted from the natural producer B. pertussis. It was found that the partially purified r-CyaA had about 4-fold lower specific hemolytic activity on sheep erythrocytes and 4-10-fold lower specific pore-forming activity in artificial planar lipid bilayers than Bp-CyaA (9) , whereas the cell-invasive activity of both proteins was equal. In the present studies, r-CyaA and Bp-CyaA were mixed at different molar ratios, so as to yield a fixed total CyaA concentration of 1 unit/ml, and the cell-invasive and hemolytic activities of the mixtures were determined. The cell-invasive AC activity was constant regardless of the molar ratio of the proteins in the mixture (Fig. 1). In contrast, the hemolytic activity of the mixture increased with the increasing proportion of the Bp-CyaA present in the mixture, yielding an upwardly concave curve. Because both cell-invasive and hemolytic activities depend on the posttranslational fatty-acylation of Lys of CyaA(14, 15) , we asked whether or not the difference in the hemolytic activities of r-CyaA and Bp-CyaA could be accounted for by differences in the chemical nature and/or location of their posttranslational modifications.


Figure 1: Comparison of cell-invasive and hemolytic activities of r-CyaA and Bp-CyaA. Toxin dilutions and/or mixtures were prepared in 50 mM Tris-HCl, pH 8.0, 8 M urea, and 2 mM EDTA. R-CyaA and Bp-CyaA were mixed at various molar ratios to obtain the final solutions at 100 units/ml (200 µg/ml) of total CyaA, and the toxin mixtures were directly diluted to 1 unit/ml (100-fold) into prewarmed suspensions of washed sheep erythrocytes (5 10^8/ml) in TNC (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM CaCl(2)). The suspensions were incubated at 37 °C. After 30 min, aliquots of the suspensions were chilled on ice and washed with cold TNE buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA). Then L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin was added to 40 µg/ml, and the suspensions were incubated at 37 °C for 10 min in order to destroy the AC toxin remaining outside the erythrocytes. Upon addition of soybean trypsin inhibitor (2-fold excess) the erythrocytes were washed again, lysed in 50 mM Tris, pH 8.0, 0.2 mM CaCl(2), 0.1% Nonidet P-40 and the adenylate cyclase enzymatic activity, which had penetrated into the erythrocytes and was protected against externally added trypsin, was measured. The extent of erythrocyte lysis was determined after 130 min of incubation by spectrophotometrically measuring the released hemoglobin in cell-free incubation supernatants at 541 nm. Detergent lysed erythrocyte suspension was used to determine the 100% lysis value.



When characterized by mass spectrometry, both native Bp-CyaA and r-CyaA matched the amino acid sequence deduced from the published nucleotide sequence of the cyaA gene (10, 17) and exhibited the following posttranslational modifications. Bp-CyaA has previously been shown to bear a single modification consisting of a palmitoylation at the -amino group of Lys(15) . As expected, this acylation was observed only when the toxin was produced in the presence of a functional CyaC protein and was not observed in Bp-proCyaA(15) . The pattern of acylation in the r-CyaA, however, differed from that observed in the B. pertussis-derived protein. Both palmitoylation (87%) and myristoylation (13%) were observed at Lys (n = 3, RSD = 23%). About 67% (n = 3, RSD = 32%) of the r-CyaA molecules were found to bear a second modification at Lys, consisting entirely of palmitoylation, with the remaining Lys residues unmodified (Fig. 2). Acylation at Lys was also strictly CyaC-dependent.


Figure 2: Most of Lys residues of r-CyaA are palmitoylated. A, CAD spectrum of the tryptic fragment of r-CyaA spanning residues Thr to Lys and containing palmitoylation at Lys. The parent ion was [M + 2H], m/z 941. For definitions of b and y ions, see (30) . B, MALDI-TOF MS spectrum of the late eluting microbore HPLC fraction containing palmitoylated Lys r-CyaA, observed at m/z 5359 and encompassing residues Asp to Gln (DIASRKGERPALTFITPLAAPGEEQRRRTKTGKSEFTTFVEIVGKQ), with a predicted average mass (protonated) of 5358. C, MALDI-TOF MS spectrum of the fraction containing unmodified Lys at m/z 5120. The predicted average mass was 5120. The peaks observed in the MALDI spectra at m/z 4284 and 8566 are from bovine ubiquitin, which was added to the samples as an internal standard for mass calibration.



Both Lys and Lys of CyaA lie in regions highly conserved among all RTX toxins. Although acylation of the two corresponding lysine residues of the homologous E. coli alpha-hemolysin (HlyA) has been observed in vitro(26) , the pattern of in vivo acylation of the naturally occurring HlyA remains unknown. Nevertheless, these data led to the suggestion that both Lys and Lys of CyaA might be acylated(26) . This, however, appears to be the case only for r-CyaA produced in E. coli and not for the naturally occurring Bp-CyaA produced by B. pertussis. An abundant tryptic fragment, corresponding to residues 861-872 of Bp-CyaA, was identified by MALDI-TOF MS and sequenced by tandem MS (Fig. 3). This fragment would not be present in tryptic digests if Lys was acylated, thereby eliminating the tryptic cleavage site. The tryptic peptide containing unmodified Lys and spanning residues Thr to Lys was not recovered by our HPLC procedures(15, 21) . A weak signal (signal/noise 5) at m/z 1882 was observed by MALDI-TOF, in an HPLC fraction corresponding to the expected elution time of the tryptic palmitoylated Lys-containing peptide. The calculated average mass for this peptide was 1882.3. The predicted HPLC behavior was based on the retention times of a synthetic acyl peptide standard (same structure as that shown in Fig. 2A) and the tryptic acyl peptide isolated from the Lys site in r-CyaA (Fig. 2A). The amount recovered from Bp-CyaA was insufficient to generate a CAD spectrum. Only one abundant Asp-N fragment was isolated from this site, observed [M + H] at m/z 5120, consistent with unmodified Lys (see the sequence given in the legend for Fig. 2). Based on the results discussed above, we estimate that Bp-CyaA palmitoylated at Lys represents less than 5% of the total protein.


Figure 3: Lys of Bp-CyaA is essentially unmodified. CAD spectrum of the unmodified fragment isolated from a tryptic digest of Bp-CyaA, which could only result from cleavage at an unmodified Lys. This fragment spans residues Ser to Lys. The parent ion was [M + 2H], m/z 679.



Because of its conservation in other RTX toxins(15, 26) , the Lys site of r-CyaA was also investigated with respect to potential modifications. As was reported for Bp-CyaA(15) , only unmodified Lys was observed.

It remains unclear why in the presence of the same CyaC protein, the CyaA is differently acylated in E. coli and in B. pertussis. One hypothesis is that acylation of AC toxin is not catalyzed by CyaC itself, but rather that it is catalyzed by an unidentified transacylase which uses acyl-ACP as substrate and CyaC as a co-factor. This hypothetical transacylase might have a slightly different specificity in E. coli and in B. pertussis. Indeed, the in vitro acylation experiments involving the HlyA and the CyaC homolog, HlyC, indicated that HlyC is not a conventional enzyme(27, 28) . In fact, stoichiometric amounts of HlyC were required for activation of proHlyA. Moreover, although acyl-ACP and HlyA could be labeled in vitro by a radioactive acyl chain, a radioactive acyl-HlyC intermediate was not observed. It is conceivable that the transacylating enzyme might have been present, as a minor component, in the partially purified preparations of HlyC, proHlyA, and/or acyl-ACP of E. coli, used for the in vitro acylation system. An alternative hypothesis is that CyaC may itself be catalyzing the acylation of CyaA, and when expressed in E. coli, its specificity may be affected by some alteration in the state of the overproduced r-proCyaA substrate.

The differential effect of altered acylation on toxin function, namely a reduction in hemolytic activity, but no effect on invasive activity, provides evidence against the hypothesis that the pore involved in hemolytic activity has a role in delivery of the catalytic domain(9, 29) . Since it appears from their invasive activities that Bp-CyaA and r-CyaA are comparable in their propensity to insert into the membrane, the differences in their acylation state must affect a subsequent step in toxin action. The most likely candidate for this functional defect is the oligomerization of CyaA molecules to form the hemolytic pore. Intoxication of target cells occurs as a linear function of AC toxin concentration(18) , indicating a monomolecular mechanism for delivery of the catalytic domain. (^3)In contrast, channel-forming and hemolytic activities are a non-linear function of toxin concentration exhibiting a cooperativity coefficient suggestive of a toxin tetramer(8, 18) . On the basis of these data, we propose that CyaA molecules in different states of oligomerization, but especially monomers, can deliver their catalytic domains into cells, whereas oligomerization of AC toxin is a prerequisite for formation of hemolytic channels.

The data suggesting that overacylated r-CyaA is selectively impaired in the formation of the CyaA channels (Fig. 1) indicate that besides being essential for the interaction of CyaA with the membrane, the acylation of CyaA may also be involved in oligomerization of the CyaA molecules and formation of CyaA channels. It remains to be determined how the excess acylation on Lys of r-CyaA can impair its channel-forming activity. Defining this mechanism will contribute to our general understanding of the role of fatty-acylation in protein-membrane and protein-protein interactions.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI18000 (to E. L. H.) and GM37537 (to D. F. H.), Institut Pasteur Grant CNRS URA D1129, the Human Science Frontier Program Organization (to A. U.), the Agence Nationale des Recherches sur le SIDA (to P. S.), and an Amgen postdoctoral fellowship (to M. H.). 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.

§
To whom correspondence should be addressed: Box 419, School of Medicine, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5945; Fax: 804-982-3830; eh2v{at}galen.med.virginia.edu.

Present address: Institute of Microbiology, Czech Academy of Sciences, CZ-142 20 Prague, Czech Republic. sebo{at}biomed.cas.cz.

(^1)
The abbreviations used are: AC, adenylate cyclase; CAD, collision-activated dissociation; Fmoc, N-9-fluorenylmethoxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS, mass spectrometry; RSD, relative standard deviation; RTX, repeat in toxin.

(^2)
S. Vemuri and T. D. Lee(1989) City of Hope, Duarte, CA. This software is currently marketed by Perkin-Elmer Sciex under the name Mac Biospec.

(^3)
A. Otero, X. B. Yi, M. C. Gray, G. Szabo, and E. L. Hewlett, unpublished data.


ACKNOWLEDGEMENTS

We thank Teresa Bishop for her help with the manuscript.


REFERENCES

  1. Confer, D. L., and Eaton, J. W. (1982) Science 217,948-950 [Medline] [Order article via Infotrieve]
  2. Hanski, E., and Coote, J. G. (1991) in Sourcebook of Bacterial Toxins (Alouf, J. E., and Freer, J. H., eds) pp. 349-366, Academic Press, New York
  3. Mock, M., and Ullmann, A. (1993) Trends Microbiol. 1,187-192 [CrossRef][Medline] [Order article via Infotrieve]
  4. Hewlett, E. L., and Maloney, N. J. (1994) in Handbook of Natural Toxins, Volume 8: Microbial Toxins (Iglewski, B., Moss, J., Tu, A. T., and Vaughan, M., eds) pp. 425-439, Marcel Dekker, New York
  5. Bellalou, J., Sakamoto, H., Ladant, D., Geoffroy, C., and Ullmann, A. (1990) Infect. Immun. 58,3242-3247 [Medline] [Order article via Infotrieve]
  6. Ehrmann, I., Gray, M., Gordon, V., Gray, L., and Hewlett, E. L. (1991) FEBS Lett. 278,79-83 [CrossRef][Medline] [Order article via Infotrieve]
  7. Rogel, A., Meller, R., and Hanski, E. (1991) J. Biol. Chem. 266,3154-3161 [Abstract/Free Full Text]
  8. Szabo, G., Gray, M. C., and Hewlett, E. L. (1994) J. Biol. Chem. 269,22496-22499 [Abstract/Free Full Text]
  9. Benz, R., Maier, E., Ladant, D., Ullmann, A., and Sebo, P. (1994) J. Biochem. (Tokyo) 269,27231-22239
  10. Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2,19-30 [Medline] [Order article via Infotrieve]
  11. Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., and Danchin, A. (1988) EMBO J. 7,3997-4004 [Abstract]
  12. Welch, R. (1991) Mol. Microbiol. 5,521-528 [Medline] [Order article via Infotrieve]
  13. Coote, J. G. (1992) FEMS Microbiol. Rev. 88,137-162
  14. Barry, E. M., Weiss, A. A., Ehrmann, I. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. S. (1991) J. Bacteriol. 173,720-726 [Medline] [Order article via Infotrieve]
  15. Hackett, M., Guo, L., Shabanowitz, J., Hunt, D., and Hewlett, E. (1994) Science 266,433-435 [Medline] [Order article via Infotrieve]
  16. Heveker, N., Bonnaffe, D., and Ullmann, A. (1994) J. Biol. Chem. 269,32844-32847 [Abstract/Free Full Text]
  17. Sebo, P., Glaser, P., Sakamoto, H., and Ullmann, A. (1991) Gene (Amst.) 104,19-24 [CrossRef][Medline] [Order article via Infotrieve]
  18. Betsou, F., Sebo, P., and Guiso, N. (1993) Infect. Immun. 61,3583-3589 [Abstract]
  19. Sakamoto, H., Bellalou, J., Sebo, P., and Ladant, D. (1992) J. Biol. Chem. 267,13598-13602 [Abstract/Free Full Text]
  20. Ladant, D., Michelson, S., Sarfati, R., Gilles, A., Predelenau, R., and Barzu, O. (1989) J. Biol. Chem. 264,4015-4020 [Abstract/Free Full Text]
  21. Gulden, P. H., Hackett, M., Addona, T., Guo, L., Walker, C. B., Sherman, N. E., Shabanowitz, J., Hewlett, E. L., and Hunt, D. F. (1995) in Mass Spectrometry in the Biological Sciences (Burlingame, A. L., and Carr, S. A., eds) pp. 281-306, Humana, Clifton, NJ
  22. Hunt, D. F., Yates, J. R., Shabanowitz, J., Winston, S., and Hauer, C. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,6233-6237 [Abstract]
  23. Hunt, D. F., Alexander, J. E., McCormack, A. L., Martino, P. A., Michel, H., Shabanowitz, J., Sherman, N., Moseley, M. A., Jorgenson, J. W., and Tomer, K. B. (1991) in Techniques in Protein Chemistry II (Villafranca, J. J., ed) pp. 441-454, Academic Press, New York _
  24. Miller, J. C., and Miller, J. N. (1984) Statistics for Analytical Chemistry , 1st Ed., pp. 33-50, Ellis Horwood, New York _
  25. Fields, G. B., and Noble, R. L. (1990) Int. J. Peptide Protein Res. 35,161-214 [Medline] [Order article via Infotrieve]
  26. Stanley, P., Packman, L. C., Koronakis, V., and Hughes, C. (1994) Science 266,1992-1996 [Medline] [Order article via Infotrieve]
  27. Issartel, J., Vassilis, K., and Hughes, C. (1991) Nature 351,759-761 [CrossRef][Medline] [Order article via Infotrieve]
  28. Hardie, K. R., Issartel, J. P., Koronakis, E., Hughes, C., and Koronakis, V. (1991) Mol. Microbiol. 5,1669-1679 [Medline] [Order article via Infotrieve]
  29. Rogel, A., and Hanski, E. (1992) J. Biol. Chem. 267,22599-22605 [Abstract/Free Full Text]
  30. Biemann, K. (1990) Methods Enzymol. 193,886-887 [Medline] [Order article via Infotrieve]
  31. Arnott, D., Shabanowitz, J., and Hunt, D. F. (1992) in Proceedings of the 40th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, Washington, D. C., May 31 to June 5 , pp. 328-329, American Society for Mass Spectrometry, Santa Fe, NM
  32. Novabiochem Technical Note 3/93 (1993) Novabiochem, San Diego, CA

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