Student Theses and Dissertations
Date of Award
1980
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Daniel Rifkin
Keywords
plasma fibronectin, polypeptide fragments, NH2-terminal sequence, gelatin-binding, thrombin cleavage, amino acid analysis
Abstract
Plasma fibronectin is a glycoprotein that consists of two polypeptide chains of approximately 250,000 and 245,000 daltons, joined by disulfide bonds located near one end of the molecule. Fibronectin was isolated from human plasma by gelatin-agarose affinity chromatography and DEAE-cellulose ion exchange chromatography. The two subunit chains were found to be similar or identical with respect to primary structure, using a one-dimensional peptide mapping technique. Purified plasma fibronectin was cleaved with human α-thrombin, and three major fragments were recovered: two large ones with molecular weights of approximately 230,000 and 235,000, and a small one with a molecular weight of 29,000. All three fragments appeared to consist of single polypeptide chains. The fragments were separated on a column of gelatin-agarose. The smaller piece was not retained, whereas the larger fragments and remaining undigested molecules were bound and could be eluted with 2 M guanidine HCl. NH2-terminal analysis was performed on intact plasma fibronectin and on the fragments using the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride technique. No NH2-terminal residue was detected in either the intact molecule or the small fragment, whereas analysis of the large fragments yielded dansyl-alanine. Treatment of the intact molecule and the small fragment with L-pyroglutamyl-peptide hydrolase resulted in the appearance of NH2-terminal alanine. These results were confirmed by automated amino acid sequence analysis, which demonstrated that the intact molecule and the 29,000 dalton fragment contained the same NH2-terminal sequence, 2- terminus of intact plasma fibronectin, whereas the large fragments constitute the carboxyl portion of the molecule. Analysis of fibronectin isolated from the conditioned medium of human embryonic fibroblast cultures yielded similar results. A 72,000 dalton, gelatin-binding polypeptide was isolated from cathepsin D digests of plasma fibronectin. This fragment was cleaved further with thrombin to produce a 43,000 dalton fragment which retained the ability to bind to gelatin and a 29,000 dalton fragment which did not. NH2-terminal analysis of the 72,000 dalton and 29,000 dalton fragments by the dansyl chloride method before and after treatment with L-pyroglutamyl-peptide hydrolase indicated that they shared the same NH2-terminal sequence, 2-terminal residues of this fragment were identical with those of the intact molecule. The 43,000 dalton fragment contained an NH2-terminal alanine residue, as monitored by dansylation. These findings indicate that the 43,000 dalton fragment, which contains one or more gelatin-binding sites of plasma fibronectin, is located adjacent to the 29,000 dalton NH2-terminal region. Both the 29,000 dalton and 43,000 dalton fragments are enriched in cysteine and half-cystine, as demonstrated by amino acid analysis. The results of this thesis research can be combined with data of other investigators to locate several sites of biological interest within the fibronectin molecule, including binding sites for collagen, fibrinogen, heparin, and cell surfaces.
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Recommended Citation
Furie, Martha B., "Studies on the Structure of Human Fibronectin" (1980). Student Theses and Dissertations. 477.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/477
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy