THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 10, Issue of May 25, pp. 6524-6529, 1983 Prrnted in U. S. A.

Solubilization, Isolation, and Immunochemical Characterization of the Major Outer Membrane Protein from Rhodopseudomonas sphaeroides*

(Received for publication, October 27, 1982)

Carolyn D. Deal$ and Samuel Kaplang From the Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent iso- lation, has been achieved by both non-detergent- and detergent-based methods. The protein was differen- tially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE- Sephadex chromatography and preparative electro- phoretic techniques. Similar to the peptidoglycan-as- sociated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immuno- chemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody di- rected against the major outer membrane protein re- acted with the three high molecular weight aggregates present in the outer membrane which we have previ- ously shown to be composed of the major outer mem- brane protein and three nonidentical small molecular weight proteins.

Studies in many of the Gram-negative bacteria have re- vealed the outer membrane to be a complex structure involved not only in maintaining the structural integrity of the cell (l), but also actively involved in such physiological roles as pore formation (2-4), mediation of nutrient transport and binding activities (5-7), and phage and colicin receptor formation (8- 10). Despite the molecular complexity of the outer membrane, there appears to be an underlying similarity among many genera studied, although subtleties in detail are well docu- mented. Many similarities have been shown to exist in the proteins found in the outer membranes of various bacteria, such as the heat-modifiable proteins (11, 12), pore-forming proteins (2 ,3) , peptidoglycan-associated proteins (13-16), and lipoproteins (17-19).

Previous studies in our laboratory (20) have reported on the initial characterization of the isolated outer membrane of Rhodopseudomonas sphaeroides from chemoheterotrophi-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of United States Public Health Service Traineeship GM-7283 from the National Institutes of Health. Present address, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

fj Supported by Research Grant GM 15590 from the National Institutes of Health. To whom all correspondence should be ad- dressed.

cally grown cells. These studies demonstrated that R. sphae- roides possesses a complex outer membrane protein array which has many of the characteristics commonly associated with other Gram-negative bacteria, but in addition this struc- ture demonstrates more complex interactions between intra- membranous proteins. These interactions center around the formation of complexes between the major outer membrane protein and each of three nonidentical, smaller molecular weight polypeptides (20). In addition, the major outer mem- brane protein was suggested to contain covalently attached lipid (20). Although there is as yet no definite proof of the function of the major outer membrane protein of R. sphae- roides, the fact that it is present as a common subunit in each of the aggregates together with its abundance would suggest an important role for it in the outer membrane. To further investigate the physical and chemical characteristics of this protein, as well as its physiological role, studies were initiated to isolate this protein in homogeneous form.

Studies involving the intracytoplasmic membrane proteins of R. sphaeroides by Cohen and Kaplan (21) have demon- strated the applicability of disrupting the membrane with the chaotropic salt guanidine thiocyanate as initially described by Moldow et al. (229, followed by its replacement by dialysis against concentrated urea solutions, to obtain proteins in solubilized aqueous form, which are then amenable to further purification procedures. This technique was suggested to be of general use in the isolation of membrane proteins. In this paper, we report the application of this method to the isolation of the major outer membrane protein of R. sphaeroides. In addition, since initial studies suggested a possible porin-re- lated function for the major outer membrane protein (20), we have applied standard techniques of isolation in the anionic detergent sodium dodecyl sulfate to the outer membrane of R. sphaeroides in order to compare the behavior of this protein to that reported for peptidoglycan-associated proteins identified in outer membranes of other genera of Gram-nega- tive bacteria (13-16).

MATERIALS AND METHODS AND RESULTS”’

DISCUSSION

While there have been reports in the literature on the separation and characterization of the cytoplasmic membrane

’ Portions of this paper (including “Materials and Methods,” “Results,” Figs. 1-6, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biochemical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-2918, cite the authors, and include a check or money order for $8.00 per set of photocopies. Full sue photocopies are also included in the microfii edition of the Journal that is available from Waverly Press.

The abbreviations used are: GuSCN, guanidine thiocyanate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

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Outer Membrane Protein Purification 6525

and outer membrane of members of the Rhodospirillaceae (20,40-42), we report here, to our knowledge, the fist isolation of an outer membrane protein from this genera and an initial effort to relate it to the outer membrane proteins identified in other Gram-negative bacteria. In addition, we have demon- strated the more general applicability of the GuSCN-urea solubilization procedure of Cohen and Kaplan (21) as utilized by Moldow et al. (22) to problems of membrane protein isolation. Although other interactions may affect polypeptide solubility, as seen with the alteration in the solubility of the 29-, 26.5, and 21.5-kDa polypeptides following lysozyme treat- ment (Fig. 2), the solubilization yields a polypeptide prepa- ration in aqueous solution which is amenable to further puri- fication procedures and characterization.

The major outer membrane protein was purified by a com- bination of DEAE-Sephadex column chromatography and preparative polyacrylamide gel electrophoresis. The proce- dure resulted in a homogeneous polypeptide preparation whose purity has been demonstrated on SDS-PAGE (Fig. 4) which allows examination by size, as well as on 8 M urea- PAGE and isoelectric focusing (Fig. 4) which allows exami- nation by charge. In addition, the preparation was homoge- neous when analyzed by the immunochemical techniques of crossed immunoelectrophoresis and Western immunoblotting of SDS-polyacrylamide gels (Fig. 5). When the isolation pro- cedure was applied to outer membranes derived from photo- heterotrophically grown cells, the resulting homogeneous polypeptide was identical with the polypeptide isolated from chemoheterotrophically grown cells by all of the above crite- ria. The further physical and chemical characterization of the isolated polypeptide will be reported.

Although examination of the chemical nature of the poly- peptide is important in defining the chemical properties of the outer membrane, in addition, we wanted to correlate our observations with those reported for other Gram-negative bacterial outer membranes. For these reasons and the initial observations suggesting a porin function for this protein (20), we examined the solubilization of the outer membrane pro- teins by the anionic detergent SDS, which has been exten- sively used by other workers (13-16) to investigate peptido- glycan-associated lipoproteins and matrix or porin proteins. In addition to being a method by which to compare the R. sphaeroides protein to peptidoglycan-associated proteins identified in other Gram-negative bacteria, this approach yielded a detergent-based method for purification of the major outer membrane protein.

The major outer membrane protein was purified by a com- bination of SDS extraction of the outer membrane at room temperature and 75 “C (see “Materials and Methods”) com- bined with preparative SDS-PAGE (Table 11). This resulted in a homogeneous polypeptide which behaved identically with the protein isolated by the GuSCN-urea procedure (Fig. 4) when examined on SDS-PAGE and on 8 M urea-PAGE and isoelectric focusing following removal of SDS. By immuno- chemical criteria, the polypeptide isolated by the SDS pro- cedure was shown to be analogous to the polypeptide isolated by the GuSCN-urea procedure (Fig. 5).

Similar to the behavior of outer membrane proteins of other Gram-negative bacteria (13-16), the solubilization of the R. sphaeroides outer membrane in SDS revealed that the major outer membrane protein was retained by the peptidoglycan fraction at room temperature, but was released during 75 “C solubilization. This suggested that the major outer membrane protein is tightly associated with, but not covalently bound to, the peptidoglycan. The noncovalent nature was addition- ally supported by the solubility of the protein in GuSCN-urea. Other outer membrane proteins are not retained significantly

by peptidoglycan with the SDS extraction conditions em- ployed. The behavior demonstrated by the major outer mem- brane protein is analogous to that reported for Protein H of Pseudomonas aeruginosa by Mizuno and Kageyama (25) and for the matrix protein of Escherichia coli by Rosenbusch (13) in that the protein is SDS-insoluble at low temperatures but SDS-soluble at higher temperatures, although the exact tem- peratures of solubilization vary with the bacterial species. By analogy to the observations in other Gram-negative bacteria (13-N), we would suggest the possibility of a porin function for the major outer membrane protein; however, the unam- biguous proof of this function may be more complicated than for other Gram-negative bacteria. If the apparent molecular weight of the three aggregates on SDS-polyacrylamide gels (20) is reflective of the in vivo molecular weight, then it could be suggested that the pore is a dimer, or multiple thereof, and composed of two heterologous subunits. Achieving the proper ratio of subunits in reconstitution studies of pore formation could be difficult due to the heterogeneous nature of the aggregates (20), as opposed to the homogeneous trimers ob- served in E. coli (43,44).

Although the apparent molecular mass of 47,000 Da on 10% SDS-PAGE is larger than that reported for the peptidoglycan- associated proteins of other genera (33,000-44,000 Da), this is due to the anomalous electrophoretic mobility in SDS-gels of this protein, as will be reported, and its molecular mass is actually closer to that of the other proteins described. Further work utilizing the antibodies directed against the major outer membrane polypeptide is being directed at investigating the interaction(s) of these polypeptides within the outer mem- brane of R. sphaeroides.

REFERENCES 1. Costerton, J. W., Ingram, J. M., and Cheng, K. J. (1974) Bacteriol.

2. Nakae, T. (1976) Biochem. Biophys. Res. Commun. 71,877-884 3. Nakae, T. (1976) J. Biol. Chem. 251,2176-2178 4. Pugsley, A. P., and Schnaitman, C. A. (1978) J. Bacteriol. 133,

5. Szmelcman, S., Schwartz, S., Silhavy, T. J., and Boos, W. (1976)

6. Bassford, P. J., Jr., and Kadner, R. J. (1977) J. Bucteriol. 132,

7. Braun, V., Hancock, R. E. W., Hantke, K., and Hartman, A.

8. Datta, D. B., Arden, B., and Henning, U. (1977) J. Bacteriol. 131,

9. Foulds, J., and Chai, T.-J. (1978) J. Bacteriol. 133, 1478-1483

Rev. 38,87-110

1181-1189

Eur. J. Biochem. 65,13-19

796-805

(1976) J. Supramol. Struct. 5, 37-58

821-829

10. Konisky, J. (1979) in Bacterial Outer Membranes: Biogenesis and Function (Inouye, M., ed) pp. 319-359, Wiley-Interscience, New York

11. DiRienzo, J. M., Nakamura, K., and Inouye, M. (1978) Annu. Rev. Biochem. 47,481-532

12. Frash, C. E., and Mocca, L. F. (1978) J. Bacteriol. 136, 1127-1134 13. Rosenbusch, J. P. (1974) J. Biol. Chem. 249,8019-8029 14. Hasegawa, Y., Yamada, H., and Mizushima, S. (1976) J. Biochem.

15. Mizuno, T. (1979) J. Biochem. 86,991-1000 16. Mizuno, T. (1981) J. Biochem. 89, 1039-1049 17. Braun, V., and Rehn, K. (1969) Eur. J. Biochem. 10,426-438 18. Nakamura, K., Pirtle, R. M., and Inouye, M. (1979) J. Bacteriol.

19. Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem.

20. Baumgardner, D., Deal, C., and Kaplan, S. (1980) J. Bacteriol.

21. Cohen, L. K., and Kaplan, S. (1981) J. Biol. Chem. 256, 5901-

22. Moldow, C., Robertson, J., and Rothfield, L. (1972) J. Membr.

23. Sistrom, W. R. (1960) J. Gen. Microbiol. 22, 778-785 24. Ding, D. H., and Kaplan, S. (1976) Prep. Biochem. 6, 61-79 25. Mizuno, T., and Kageyama, M. (1979) J. Biochem. 86,979-989

80, 1401-1409

137, 595-604

256, 3125-3129

143, 265-273

5908

Biol. 10, 137-152

by guest on June 18, 2018http://w

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6526 Outer Membrane Protein Purification

26. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 27. Jovin, T., Chrambach, A,, and Naughton, M. A. (1964) Anal.

28. Righetti, P. G., and Drysdale, J. W. (1974) J. Chromatogr. 98,

29. Higgins, R. C., and Dahmus, M. E. (1979) Anal. Biochem. 93,

30. Shepherd, W. D., and Kaplan, S. (1978) J. Bacteriol. 135, 656-

31. Watt, R. M., Herron, J., and Voss, E. M. (1980) Mol. Immunol.

32. Burnette, W. N. (1981) Anal. Biochem. 112,195-203 33. Kranz, R. G., and Gennis, R. B. (1982) Anal. Biochem. 127,

34. Hunter, W. M., and Greenwood, F. C. (1962) Nature (Lond.) 194,

35. Munkres, K. D., and Richards, F. M. (1965) Arch. Biochem.

Biochem. 9,351-369

271-321

257-260

667

17, 1237-1243

247-257

495-496

Biophys. 109,466-479

Biochem. 93,153-157

Bacteriol. 133, 306-319

biol. Lett. 7,349-353

36. Henderson, L. E., Oroszlan, S., and Konigsberg, W. (1979) Anal.

37. Smyth, C. J., Siegel, J., Salton, M. R. J., and Owen, P. (1978) J.

38. Brown, A. E., Calder, K., and Lascelles, J. (1980) FEMS Micro-

39. Kranz, R. G., and Gennis, R. B. (1982) J. Bacteriol. 150, 36-45 40. Collins, M. L., and Niederman, R. A. (1976) J. Bacteriol. 126,

41. Guillotin, J., and Reiss-Husson, F. (1975) Arch. Microbiol. 105, 269-275

42. Oelze, J., Golecki, J. R., Kleinig, H., and Weickessi, J. (1975) Antonie Leeuwonhoek J. Microbiol. Serol. 41,273-286

43. Palva, E. T., and Randall, L. L. (1978) J. Bacteriol. 133,279-286 44. Nakae, T., Ishii, J., and Tokunaga, M. (1979) J. Biol. Chem. 254,

1316-1325

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Outer Men SUPPLEMENTARY IIRTERIAL TO:

SOLUBILIZATION, ISOLATION AND IWMUNOCHEVICAL CHARACTERIZATION OF THE M U O R OUTER HElBRbNE PROTEIN FROM RHOWPSEUWMONAS

SPHAEROIDES.

Carolyn D. Deal and Samuel Kaplan

MATERIALS AND METHODS

Outer Mcrnbrane Isolation Outer membrane fractions were prepared by the method of Dlnq and Kaplan, 124, see also 201.

Throcyanate ICuSCN) Hiqhly purifled Outer membranes were Extraction and Solubrliration of Outer n m b r m e s by Guanidine

Tialyred exrensiveIy aga ins t qlass distilled H z 0 and lyophl-

scetone/aerhanol 17:2, v/v) accordinq t o Cohen and Kaplan 1211. lrred. Phosphollplds were extracted from outer membranes with

The 1yophllLzed membrane pcoteln I d s CeSuspendFd by 80nlCation in

Cine, 5 nM EDTA and 5 mM 2-mer~dpt0ethanol at a proteln Concen- 1 M GuSCN contarnlnq 50 mH sodium phosphate pH 7 . 4 , 10 mM gly-

tration Of 5 mq/ml and strrred for 30 rnln at room temperature. The Solution was dlalyred agalnat two changes 150:l v/v. 10 to

the 1nltla1 mlubillzatlon in GuSCN. The insoluble protein was 12 h each) of 711 urea mntslninq the same buffer a d aqtnts as in

pelleted by centrifuqatlon at 150,OOOxg for 3 h (Beckman 50 Ti rotor1 at 1.C.

temperature in 511 GuSCN containing the same buffers and agents as The resulting pellet Ya8 solubilized for 30 nin at cwm

ni/ml. The solution was dmlyred aqsinat 711 urea containing lo in the initial solubilrration at a protein mncentration of 5

m TriS pH 7.2 and 5 mM 2-mercaptoethanol. This dialysis. as well as all subsequent handllnq, was carried out at 4'C. Pollor-

t i o n at 150,OOOxq for 3 h. The urea soluble supernatant was used Inq dialysis, the insoluble proteins were removed by centrifuqa-

immediately for subsequent fractionatron.

I.~sozyme Treatment Samples suspended I n 0.1 n sodium phosphate buffer p~ 7.6 were treated with lysozyme at a enzyme to protein ratlo Of 1:50 (It/Yt) for 30 min 4t 3O'C. The fCaCtiOn Vas then

GuSCN as detalled abOve. dralyred aqainst distilled HIO, lYoDhilized, and extracted with

Solubilization of Outer 11embrmeJ by SDS The preparation Of SDS soluble and insoluble proteins was based on the method of niruno

membranes were extracted in 2 1 5 0 5 , 10 rn Tria pH 7.8 for 30 min (251 wlth the following mcddlficationa. Hiqhly purified outer

at 27'c. Insoluble polypeptldes were removed by centrifugation at 150,OOOxq for 1 h (Beckman 50 Ti rotor1 at 15.C. The result-

75.C for varyinq amounts Of tine. The insoluble polypeptides Inq pellet was extracted in 2) SDS containing the sane hffers at

were removed by centrifuqatlon as abOve.

Analytical POIyacrYlamzde Gel EleCtrOPhoTesi8 10% SDS - poly- acrylamide Slab qels ~120x160r0.8mrn) were prepared accordinq to the method Of Lacmli 126). Prior to electrophoresis samples were solubillzed Ln 28 SDS, 62.5 mll TriS pH 6.8, 2% 2-mercapto- ethanol, 10% qlycerol and 0.011 bromophenol blue for 15 mi" at 27'C or for 7 min at 75'C. After electrophoresis qcls were stained and destained as previously described 120).

SCruCCed accordinq t o the method of JOvin (271 from a Stock solu- 8 n urea polyacrylamide 9 t h (120x160x0.8mm) were mn-

tion of 30% amylamide, 0 . 8 8 N, N'-methylcnebiaacrylanide. The Stackinq qel was 3 ) acrylamrde and the separating qel was 6a

qlycine and 5 mbl 2-mercaptoethanol. Electrophoresis was carried acrylamrde in 8 I urea. Samples were dissolved in 8 n urea, 1%

out at 240 V at 4.C utilirrnq the running buffers described (211.

conducted as described previously 121) . Upon mmplctlon of focusrnq, the pH gradient established in the qe1 was determined by SlLclnq an unused lane into 5 m Slices and measurinq the pH of each slice after soskrnq overniqht in deqassed 2% NaC1. The gels were rinsed rn 2-3 changes of 108 trichloroacetic acid to remove ampholines and then stained for protein wlth Coomassie Rrillxant Blue by thP method of Riqhetti and Dryadale (281.

Isoelectric focusinq in 5% polyacrylamide slob gels was

Synthesis Of Guanidine Thiocyanate Guanidine thiocyanate was syntheslred accordlnq to the method of COhen and Kaplan (21).

rtbrane Protein Purification -

opposed to delipidated Chromatophore protelns which ulth minor exceptions are nonselectively 901Uble in CuSCN 1211 the outer membrane proteins exhibit a differential solubilltv'in GuSCN rlth several being insoluble without prror treatment IFlq. 11.

..... ",..

obtained following the Solubilization protocol (Fig. 1, lanes 2 (FiQ. 1, lane I1 can be accounted f& in the three fractions

3 . 4 1 . This procedure results in recovery of 21% Of the startin; outer membrane protein i n the urea Soluble fraction obtained from

ately used for further purification procedures. the 511 GuSCN solubilization (Table 11. This fraction vas imedi-

I c - "- 3 4

Zf

2 '

behavior of Several Of the 5 1 GuSCN-urea insoluble polypeptldes. could be altered by lysozyme treatment Of the 1 I! GuSCN rnsoluble proteins but prior to solubilization with 5 I GuSCN as detailed under .Materia19 and Uethods". In particular the 21.5, 26.5 and 29 kilodalton polypeptldes which were prevrously insoluble under these mnditions were soluble follovlnq lysozyme treatment (Fiq.

possibly the 47 kilodslton polypeptide with rhlch they interact 2, lanes 3 and 4 ) . Thls suggests Chat these polypeptxdes, and

(201. could form an association with the peptidoqlycan.

Additionally, it was observed (Pi". 2, lanes 3-61 that the

47 .

t

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6528 Outer Membrane Protein Purification

brsted in i n urea contbininq 10 ill Tris pi 1.2, 5 mn 2-&- Captoethan01. After elution with 2 column VOlumCS Of buffCC at a flor rate of 15 m l h , the EOlumI) was eluted with a 0-0.5 U NaC1 aradicnt in 1 n urea mntsinina the same buffer. 2.0 m 1

achieved on preparative 8n urea pH8.7 polyacrylamide g e l s as Final purification of the major Outer membrane protein was

described under Watecials and nethods. and resulted in a final yreld of 98 based on the initla1 protein [Table I1 The polypep- tide was s h w n to be horwgeneous rhtn eramned On io$ SDS poly-

pn 8.7 polyacrylamide gel e1ectrophore;ia (Fig. 4 . l anes 3 and acrylamlde gel elFCtrophorFsIs IFig. 4 lanes 1 and 21, 8 M urea

1 1 , and by xaoelecrrio focuslnq (PI-4.0 in 8 n urea polyacryl- anide gels IF lg . 1, lanes 5 and 61. I n addition. the polypeptide was homqeneous by innunochemical procedures.

Table I

Recovery Of 9. sehaeroides outer membrane protelna and w t a . follorinq CltraEtlOn and solublliration with GuSCN

w $

Outer membrane 43 100

Delipidated protein 41.2 96

1 II GuSCN extracted-urea 16.6 39 SOlUhle protein

5 n GuSCN extracted-urea 8.8 21 soluble protein

Urea-insoluble protein 10.1 25

Protein eluting at 0.2 n NaC1 from DEAE-Sephadex 5.5 13

Through urea pH 8.7-PAGE 1 9

preparation containlnq the major outer membrane protein. Final The 15.C SDS soluble fraction represented a highly enCLched

purification was achieved on preparat1VC SDS polyacrylamide gel8 as deacribed under -Materials and Methods.. The mlvoeatide

polypeptlde kso1at;d by CuSCN urea 801ub11izat10n see'fxgu;e~(. This indicated that the two preparations represented isolation of the same polypeptlde and therefore m u l d be used Interchangeably

necessary for the technique to be employed. ~n further studies depending on the state of the polypeptlde

I~unOEhemiEaI AnalYBiB Of the Outer nembrane and najor Outer nembrane Protein The techniques of Crossed immnOeleCtrophoresi8 ICIFI and Western imnunohlotting have been pre~lously applied to the study of membrane proteins 133.37.38. 39). Initially r e wished to develoo these svstems to afford another criterion of

antisera was additionally revealed by inmunoblotting Of SDS poly- The mnospecificity Of the major outer membrane protein

acrylamide gels according to the method of Burdette as described under .Materials and Methods: As shown in Figure 5, lane 2, blotting of an arter membrane sample with antibody prepared against the major Outer membrane protezn resulted in a reaction with only the major outer membrane protein. Th18 was in mntrast to the outer membrane antibody which react& rlth the hulk of the bands separated on the SDS-polyacrylamide gel IFig, 5, l ane 11

peptides to the nitrocellulose by the method employed. Lanes 3 and demonstrated the transfer of a l l of the outer membrane poly-

and 1 Of Fiaure 5 demnatrated the rtactivitv of the Outer mem- brane protein antisera with both the urea pu;ifred and the SDS purified major outer membrane polypeptide, therefore indicating that the two preparations -"rain antigentically similar mmpon- ents. Taken as a whole. our results demonstrate that the poly- peptides purified by the methods reported above are each innuno- chemically homqeneous preparattons whlch are cross-reactive with each Other.

1 2 3 4

- c

FlgUre 5 AYtOradiOgcam Of innunoblots Of Outer membrane and isolated major outer menhrme proteln analyzed on SDS-PAGE.

sera and major outer membrane protern antisera respectively: 3 Lanes: 1 and 2. Outer membrane probed with outer membrane anti-

and 1. Isolated outer .Dembrane protein from SDS soluhlliration

outer membrane protein antisera. Lanes 1 and 2 contained 20 ug and GuSCN-urea Solubilfratron reapectlvely, probed with malor

of proteln; lanes 3 and 1, 1 iq protein.

protein by polyacrylamide qel electrophoresis. Lanes: 1 and 2. Figure 1 Examination of the purtfied major Outer membrane

8.1 -PAGE, 10 and 25 i;g respectively: 5 and 6. isoelectric 10% SDS-PAGE. 10 and 25 uq respectively: 3 and 4 . 8 U urea pH

focusino sel (pH 3-5.51, 10 and 25 LO respectively.

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Outer Membrane Protein Purification Aqqreqate Formation of the Major Outer Membrane Protein We have p ~ e v ~ o u s l y shown by f a dimensional polyacrylamide gel electro- phoresis 1201 that the major Outer nembranc protein forms aqqre- qates with each of three nonidentical smaller molecular reiqht polypeptides 121.5.26.5. and 29 kilodaltons). These hiqh m l e - cular weiaht a94reoateb 168, 73. and 7 5 kilodaltonsl are stable ( ~ u r ~ n q SOL sol6hiliration at mom temperature prior to electro- phoresra 1Fiq. 6, lane 1) but are drssociated by SDS solubilize- L l o n at 75.C (Flq. 6, l ane 21. AS described above, me have iso-

specific antibody directed against that protein. Outer membrane lated the major Outer membrane protein and have generated mono-

samples solubllired at room temperature and at 75.C were electro- phoresed On SDP 108 polyacrylamide gels, then transferred to

prorern antibody as described under .Materials and Methods.. The nitrocellulose for immnoblottinq with the major Outer membrane

antibody reacted with the hiqh molecular weight sqqreqates in the unheated sample (Fig. 6, lane 31 and rlth the major outer new hrane proteln in the heated Sample IFiq. 6. lane 4 ) , thus unmbiquously denanstrating the presence of the major outer membrane protein in the three hiqh molecular aggregates in the unheated sample. The reaction of the antibody with the unheated

have 1solated is the ms3or Outer membrane protein and that it and heated outer membrane samples revealed that the protein we

demonstrated the aqqreqation properties we originally observed by ta-dimensional plyacrylanide gel electrophoresis and was the cornm~n Subunit in the three hiqh m~lecular relght aqgreqates.

Recovery of the major outer membrane protein by SDS solUbillzat10n and purification.

1

75

68 72

4 7

Figure 6 SDS polyacrylamide electrophoresis of outer membrane solubilized at room tenperatwe and 75.C and autoradiogram of an

membrane solubilized at room temperature and 75.C respectively: imunoblot Of the gel. Lanes: 1 and 2, SDS-PAGE Of outer

bilized at rmm tenperatwe and 75 ' C rcspcctive1y probed with 3 and 4 , aut0re.dioqr.D. Of 1mmunoblot of outer membrane solu-

major outer membrane protein antisera. Lanes 1 and 2 contained 50 u q protein: lane 3 , 20 - 1 and lane 4 . 4 u q protein.

6529

75.C SDS soluble fraction through 4.2 preparaclve SDS-PAGE

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C D Deal and S Kaplanmembrane protein from Rhodopseudomonas sphaeroides.

Solubilization, isolation, and immunochemical characterization of the major outer

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