HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Furie, B. C. Articles by Furie, B. Search for Related Content PubMed PubMed Citation Articles by Furie, B. C. Articles by Furie, B. Social Bookmarking                      What's this?

JBC, Vol. 254, Issue 24, 12521-12530, Dec, 1979

Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin

B. C. Furie, M. Blumenstein and B. Furie

The metal binding sites of a gamma-carboxyglutamic acid-rich fragment derived from bovine prothrombin were examined using paramagnetic lanthanide ions to evaluate the role of gamma-carboxyglutamic acid resideus in metal binding. A gamma-carboxyglutamic acid-rich peptide, fragment 12-44, was isolated from a tryptic digest of prothrombin. Using 153Gd(III), fragment 12-44 was found to contain one high affinity metal binding site (KD = 0.55 microM) and four to six lower affinity metal binding sites (KD approximately 4 to 8 microM). The S-carboxymethyl derivative of fragment 12-44, in which the disulfide bond in fragment 12-44 was reduced and alkylated, contained no high affinity metal binding site and four or five lower affinity sites (KD = 8 microM). The effects of paramagnetic lanthanide ions on fragment 12-44 and its S-carboxymethyl derivative were studied by natural abundance 13C NMR spectroscopy. The 13C NMR spectrum of fragment 12-44 was recorded at 67.88 MHz and the resonances were assigned by comparison to the chemical shift of carbon resonances of amino acids and peptides previously studied. The proximity between bound metal ions and carbon atoms in fragment 12-44 was estimated using Gd(III), based upon the strategy that the magnitude of the change in the transverse relaxation rate of resonances of carbon nuclei induced by bound metal ions is related in part to the interatomic distances between bound metal and carbon nuclei. Titration of fragment 12-44 with Gd(III) resulted in the selective broadening of the gamma-carboxyl carbon, C gamma, C beta, and C alpha resonances of gamma-carboxyglutamic acid, and the C epsilon of the arginines. S-Carboxymethyl fragment 12-44, which lacked the high affinity metal binding site, showed markedly decreased perturbation of the C epsilon of the arginine residues upon titration with Gd(III). These studies indicate that gamma-carboxyglutamic acid residues in prothrombin fragment 12-44 participate in metal liganding. A high affinity metal binding site in fragment 12-44 is in close proximity of Arg 16 and Arg 25 and is stabilized by the disulfide bond. On the basis of these data, a model of the metal binding sites is proposed in which the high affinity site is composed of two gamma-carboxyglutamic acid residues which participate in intramolecular metal-dependent bridging of two regions of the polypeptide chain. The lower affinity metal binding sites, formed by single or paired adjacent gamma-carboxyglutamic acid residues, then may participate in intermolecular metal-dependent protein . protein or protein . membrane complex formation.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. M. Monroe, M. Hoffman, and H. R. Roberts Platelets and Thrombin Generation Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1381 - 1389. [Abstract] [Full Text] [PDF]

				S. J. Freedman, M. D. Blostein, J. D. Baleja, M. Jacobs, B. C. Furie, and B. Furie Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX J. Biol. Chem., July 5, 1996; 271(27): 16227 - 16236. [Abstract] [Full Text] [PDF]