Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jun 15;316(Pt 3):825–831. doi: 10.1042/bj3160825

SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

C Sagné 1, M F Isambert 1, J P Henry 1, B Gasnier 1
PMCID: PMC1217424  PMID: 8670158

Abstract

The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.

Full Text

The Full Text of this article is available as a PDF (493.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apps D. K., Pryde J. G., Phillips J. H. Cytochrome b561 is identical with chromomembrin B, a major polypeptide of chromaffin granule membranes. Neuroscience. 1980;5(12):2279–2287. doi: 10.1016/0306-4522(80)90143-8. [DOI] [PubMed] [Google Scholar]
  2. Erickson J. D., Eiden L. E., Hoffman B. J. Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10993–10997. doi: 10.1073/pnas.89.22.10993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Giraudat J., Roisin M. P., Henry J. P. Solubilization and reconstitution of the adenosine 5'-triphosphate dependent proton translocase of bovine chromaffin granule membrane. Biochemistry. 1980 Sep 16;19(19):4499–4505. doi: 10.1021/bi00560a018. [DOI] [PubMed] [Google Scholar]
  4. Hennessey J. P., Jr, Scarborough G. A. An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein. Anal Biochem. 1989 Feb 1;176(2):284–289. doi: 10.1016/0003-2697(89)90310-2. [DOI] [PubMed] [Google Scholar]
  5. Huang K. S., Bayley H., Liao M. J., London E., Khorana H. G. Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments. J Biol Chem. 1981 Apr 25;256(8):3802–3809. [PubMed] [Google Scholar]
  6. Isambert M. F., Gasnier B., Botton D., Henry J. P. Characterization and purification of the monoamine transporter of bovine chromaffin granules. Biochemistry. 1992 Feb 25;31(7):1980–1986. doi: 10.1021/bi00122a012. [DOI] [PubMed] [Google Scholar]
  7. Isambert M. F., Gasnier B., Laduron P. M., Henry J. P. Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin. Biochemistry. 1989 Mar 7;28(5):2265–2270. doi: 10.1021/bi00431a044. [DOI] [PubMed] [Google Scholar]
  8. Krejci E., Gasnier B., Botton D., Isambert M. F., Sagné C., Gagnon J., Massoulié J., Henry J. P. Expression and regulation of the bovine vesicular monoamine transporter gene. FEBS Lett. 1993 Nov 29;335(1):27–32. doi: 10.1016/0014-5793(93)80432-t. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Landon Cleavage at aspartyl-prolyl bonds. Methods Enzymol. 1977;47:145–149. doi: 10.1016/0076-6879(77)47017-4. [DOI] [PubMed] [Google Scholar]
  11. Liu Y., Peter D., Roghani A., Schuldiner S., Privé G. G., Eisenberg D., Brecha N., Edwards R. H. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell. 1992 Aug 21;70(4):539–551. doi: 10.1016/0092-8674(92)90425-c. [DOI] [PubMed] [Google Scholar]
  12. Llinás M., De Marco A., Lecomte J. T. Proton magnetic resonance study of crambin, a hyperstable hydrophobic protein, at 250 and 600 MHz. Biochemistry. 1980 Mar 18;19(6):1140–1145. doi: 10.1021/bi00547a016. [DOI] [PubMed] [Google Scholar]
  13. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  14. Nelson N. Organellar proton-ATPases. Curr Opin Cell Biol. 1992 Aug;4(4):654–660. doi: 10.1016/0955-0674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  15. Perin M. S., Fried V. A., Slaughter C. A., Südhof T. C. The structure of cytochrome b561, a secretory vesicle-specific electron transport protein. EMBO J. 1988 Sep;7(9):2697–2703. doi: 10.1002/j.1460-2075.1988.tb03123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rabilloud T., Carpentier G., Tarroux P. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis. 1988 Jun;9(6):288–291. doi: 10.1002/elps.1150090608. [DOI] [PubMed] [Google Scholar]
  17. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  18. Scherman D., Jaudon P., Henry J. P. Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine. Proc Natl Acad Sci U S A. 1983 Jan;80(2):584–588. doi: 10.1073/pnas.80.2.584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schuldiner S., Shirvan A., Linial M. Vesicular neurotransmitter transporters: from bacteria to humans. Physiol Rev. 1995 Apr;75(2):369–392. doi: 10.1152/physrev.1995.75.2.369. [DOI] [PubMed] [Google Scholar]
  20. Smith A. D., Winkler H. A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem J. 1967 May;103(2):480–482. doi: 10.1042/bj1030480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stern-Bach Y., Greenberg-Ofrath N., Flechner I., Schuldiner S. Identification and purification of a functional amine transporter from bovine chromaffin granules. J Biol Chem. 1990 Mar 5;265(7):3961–3966. [PubMed] [Google Scholar]
  22. Stoffel W., Hillen H., Schroeder W., Deutzmann R. Primary structure of the C-terminal cyanogen bromide fragments II, III and IV from bovine brain proteolipid-apoprotein. Hoppe Seylers Z Physiol Chem. 1982 Aug;363(8):855–864. doi: 10.1515/bchm2.1982.363.2.855. [DOI] [PubMed] [Google Scholar]
  23. Stoll V. S., Blanchard J. S. Buffers: principles and practice. Methods Enzymol. 1990;182:24–38. doi: 10.1016/0076-6879(90)82006-n. [DOI] [PubMed] [Google Scholar]
  24. Viitanen P., Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification, reconstitution, and characterization of the lac permease of Escherichia coli. Methods Enzymol. 1986;125:429–452. doi: 10.1016/s0076-6879(86)25034-x. [DOI] [PubMed] [Google Scholar]
  25. Vincent M. S., Near J. A. Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla. Mol Pharmacol. 1991 Dec;40(6):889–894. [PubMed] [Google Scholar]
  26. Wakefield L. M., Cass A. E., Radda G. K. Isolation of a membrane protein by chromatofocusing: cytochrome b-561 of the adrenal chromaffin granule. J Biochem Biophys Methods. 1984 Sep;9(4):331–341. doi: 10.1016/0165-022x(84)90017-4. [DOI] [PubMed] [Google Scholar]
  27. Wouters W., Van Dun J., Leysen J. E., Laduron P. M. Photoaffinity probes for serotonin and histamine receptors. Synthesis and characterization of two azide analogues of ketanserin. J Biol Chem. 1985 Jul 15;260(14):8423–8429. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES