Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Oct 1;99(4):1405–1409. doi: 10.1083/jcb.99.4.1405

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody

PMCID: PMC2113325  PMID: 6207181

Abstract

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV- specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

Full Text

The Full Text of this article is available as a PDF (783.1 KB).

Selected References

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

  1. Bächinger H. P., Fessler L. I., Fessler J. H. Mouse procollagen IV. Characterization and supramolecular association. J Biol Chem. 1982 Aug 25;257(16):9796–9803. [PubMed] [Google Scholar]
  2. Fitch J. M., Gibney E., Sanderson R. D., Mayne R., Linsenmayer T. F. Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen. J Cell Biol. 1982 Nov;95(2 Pt 1):641–647. doi: 10.1083/jcb.95.2.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fitch J. M., Mayne R., Linsenmayer T. F. Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule. J Cell Biol. 1983 Sep;97(3):940–943. doi: 10.1083/jcb.97.3.940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. GROSS J. THERMAL DENATURATION OF COLLAGEN IN THE DISPERSED AND SOLID STATE. Science. 1964 Feb 28;143(3609):960–961. doi: 10.1126/science.143.3609.960. [DOI] [PubMed] [Google Scholar]
  5. Gross J. Collagen biology: structure, degradation, and disease. Harvey Lect. 1974;68:351–432. [PubMed] [Google Scholar]
  6. Highberger J. H., Corbett C., Gross J. Isolation and characterization of a peptide containing the site of cleavage of the chick skin collagen alpha 1[I] chain by animal collagenases. Biochem Biophys Res Commun. 1979 Jul 12;89(1):202–208. doi: 10.1016/0006-291x(79)90964-1. [DOI] [PubMed] [Google Scholar]
  7. Hyashi T., Curran-Patel S., Prockop D. J. Thermal stability of the triple helix of type I procollagen and collagen. Precautions for minimizing ultraviolet damage to proteins during circular dichroism studies. Biochemistry. 1979 Sep 18;18(19):4182–4187. doi: 10.1021/bi00586a022. [DOI] [PubMed] [Google Scholar]
  8. Kühn K., Wiedemann H., Timpl R., Risteli J., Dieringer H., Voss T., Glanville R. W. Macromolecular structure of basement membrane collagens. FEBS Lett. 1981 Mar 9;125(1):123–128. doi: 10.1016/0014-5793(81)81012-5. [DOI] [PubMed] [Google Scholar]
  9. Linsenmayer T. F., Fitch J. M., Schmid T. M., Zak N. B., Gibney E., Sanderson R. D., Mayne R. Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs. J Cell Biol. 1983 Jan;96(1):124–132. doi: 10.1083/jcb.96.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mayne R., Sanderson R. D., Wiedemann H., Fitch J. M., Linsenmayer T. F. The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies. J Biol Chem. 1983 May 10;258(9):5794–5797. [PubMed] [Google Scholar]
  11. Mayne R., Wiedemann H., Dessau W., Von der Mark K., Bruckner P. Structural and immunological characterization of type IV collagen isolated from chicken tissues. Eur J Biochem. 1982 Aug;126(2):417–423. doi: 10.1111/j.1432-1033.1982.tb06796.x. [DOI] [PubMed] [Google Scholar]
  12. Mayne R., Wiedemann H., Irwin M. H., Sanderson R. D., Fitch J. M., Linsenmayer T. F., Kühn K. Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing. J Cell Biol. 1984 May;98(5):1637–1644. doi: 10.1083/jcb.98.5.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mayne R., Zettergren J. G. Type IV collagen from chicken muscular tissues. Isolation and characterization of the pepsin-resistant fragments. Biochemistry. 1980 Aug 19;19(17):4065–4072. doi: 10.1021/bi00558a025. [DOI] [PubMed] [Google Scholar]
  14. Ryhänen L., Zaragoza E. J., Uitto J. Conformational stability of type I collagen triple helix: evidence for temporary and local relaxation of the protein conformation using a proteolytic probe. Arch Biochem Biophys. 1983 Jun;223(2):562–571. doi: 10.1016/0003-9861(83)90621-5. [DOI] [PubMed] [Google Scholar]
  15. Sage H., Pritzl P., Bornstein P. Susceptibility of type V collagen to neutral proteases: evidence that the major molecular species is a thrombin-sensitive heteropolymer, [alpha 1(V)]2 alpha 2(V). Biochemistry. 1981 Jun 23;20(13):3778–3784. doi: 10.1021/bi00516a017. [DOI] [PubMed] [Google Scholar]
  16. Sakai T., Gross J. Some properties of the products of reaction of tadpole collagenase with collagen. Biochemistry. 1967 Feb;6(2):518–528. doi: 10.1021/bi00854a021. [DOI] [PubMed] [Google Scholar]
  17. Sariola H., Timpl R., von der Mark K., Mayne R., Fitch J. M., Linsenmayer T. F., Ekblom P. Dual origin of glomerular basement membrane. Dev Biol. 1984 Jan;101(1):86–96. doi: 10.1016/0012-1606(84)90119-2. [DOI] [PubMed] [Google Scholar]
  18. Schmid T. M., Linsenmayer T. F. Denaturation-renaturation properties of two molecular forms of short-chain cartilage collagen. Biochemistry. 1984 Jan 31;23(3):553–558. doi: 10.1021/bi00298a024. [DOI] [PubMed] [Google Scholar]
  19. Timpl R., Wiedemann H., van Delden V., Furthmayr H., Kühn K. A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem. 1981 Nov;120(2):203–211. doi: 10.1111/j.1432-1033.1981.tb05690.x. [DOI] [PubMed] [Google Scholar]
  20. Yurchenco P. D., Furthmayr H. Self-assembly of basement membrane collagen. Biochemistry. 1984 Apr 10;23(8):1839–1850. doi: 10.1021/bi00303a040. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES