Conformational change precedes the formation of multimeric fibronectin.
Plasma fibronectin incubated with a low concentration of SH reagent under physiological conditions without cells formed a multimer which retained the ability of heparin-binding and cell-binding but lost gelatin affinity [Sakai, K., Fujii, T., and Hayashi, T. (1994) J. Biochem. 115, 415-421]. The conformation of the multimeric fibronectin, as observed by ultraviolet circular dichroism and fluorescence spectroscopy was different from that of dimeric plasma fibronectin. Monitoring the change in ellipticity indicated that conformational change was mostly accomplished within the first 3 h of incubation with 0.5 mM dithiothreitol at 37 degrees C. In contrast, multimers became detectable after 4 h of incubation. The results indicate that the overall reaction of multimerization of plasma fibronectin consists of two steps: the initial step of conformational change of dimeric fibronectin, and the later polymerization step of the polypeptide in an altered conformation. The initial step, involving the conformational change of fibronectin, depended on temperature: it proceeded at 37 degrees C but not at 25 degrees C. In contrast the second step took place at 25 degrees C at a low, yet significant rate. Proteolytic susceptibility of the fibronectin to thermolysin or m-calpain changed within 3 h of incubation with dithiothreitol at 37 degrees C in accordance with the conformational change detected by circular dichroism. Namely, the fibronectin in an altered conformation appeared to be less susceptible to thermolysin, but more susceptible to m-calpain. The changes in enzymatic susceptibilities tended to be localized in the amino- and carboxyl-terminal regions, which are consistent with the implications from the spectroscopic analysis.
Nippi Research Institute of Biomatrix, Adachi-ku, Tokyo.
K
K
Sakai
T
T
Fujii
T
T
Hayashi
D000818:Animals; D002154:Calpain / Q000494:pharmacology; D002417:Cattle; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000097:blood / Q000737:chemistry* / Q000187:drug effects; D006358:Hot Temperature; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology; D013696:Temperature; D013820:Thermolysin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D005353:Fibronectins; D013439:Sulfhydryl Reagents; D014357:Trypsin; D002154:Calpain; D013820:Thermolysin; D004229:Dithiothreitol