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Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX

Astermark, Jan LU ; Bjork, Ingemar; Öhlin, Ann-Kristin LU and Stenflo, Johan LU (1991) In Journal of Biological Chemistry 266(4). p.2430-2437
Abstract
Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic... (More)
Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bjork, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
266
issue
4
pages
2430 - 2437
publisher
ASBMB
external identifiers
  • pmid:1989994
ISSN
1083-351X
language
English
LU publication?
yes
id
e04282e5-0387-4ff9-9fa6-87142a444764 (old id 1105916)
alternative location
http://www.jbc.org/cgi/reprint/266/4/2430
date added to LUP
2008-08-04 14:03:25
date last changed
2016-04-15 20:16:34
@article{e04282e5-0387-4ff9-9fa6-87142a444764,
  abstract     = {Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bjork, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.},
  author       = {Astermark, Jan and Bjork, Ingemar and Öhlin, Ann-Kristin and Stenflo, Johan},
  issn         = {1083-351X},
  language     = {eng},
  number       = {4},
  pages        = {2430--2437},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX},
  volume       = {266},
  year         = {1991},
}