Vitamin K dependent in vitro production of prothrombin

Swanson, James R.; Suttie, John W.

doi:10.1021/bi00266a044

Cited by 31 publications

(22 citation statements)

References 30 publications

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“…We tested whether the VKS peptide affected VKD propeptide binding by assessing its ability to disrupt VKD protein-carboxylase association. In the absence of vitamin K, tight complexes between carboxylase and propeptide-VKD precursor accumulate in vivo (28,(35)(36)(37). We isolated such a preformed propeptide fIX-carboxylase complex by immobilization on anti-fIX Ab resin, then incubated the complex with various peptides, and monitored for carboxylase release from the propeptide fIX-anti fIX Ab resin (Table I).…”

Section: Free Vks Peptide Does Not Alter Carboxylase-vkd Protein Assomentioning

confidence: 99%

Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

Pudota

Hommema

Hallgren

et al. 2001

Journal of Biological Chemistry

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The vitamin K-dependent (VKD) carboxylase converts clusters of Glu residues to ␥-carboxylated Glu residues (Glas) in VKD proteins, which is required for their activity. VKD precursors are targeted to the carboxylase by their carboxylase recognition site, which in most cases is a propeptide. We have identified a second tethering site for carboxylase and VKD proteins that is required for carboxylase activity, called the vitamin K The vitamin K-dependent (VKD) 1 or ␥-carboxylase converts Glus to ␥-carboxylated Glus (Glas) in VKD proteins as they transit through the endoplasmic reticulum (1, 2). Most of the VKD proteins are secreted out of the cell, and carboxylation of their Gla domain confers the ability to bind phospholipid bilayers, where these proteins exert their effects. Carboxylation is thus required for the biological activity of VKD proteins, which function in hemostasis, calcium homeostasis, and growth control. In addition, a novel subset of mammalian VKD proteins with potential functions in signal transduction has recently been identified by sequence homology (3, 4). Unlike the other VKD proteins, these proteins apparently have a single-pass transmembrane domain with the extracellular domain containing the predicted carboxylated region. Inhibition of VKD protein activities forms the basis of anticoagulant therapies with warfarin and coumadin, in which the carboxylation of hemostatic VKD proteins, as well as the other VKD proteins, is reduced by limiting the supply of vitamin K cofactor to the carboxylase.Although the carboxylase was first identified in mammals, carboxylase homologs and activity have been found in fish, the fish-hunting cone snails of the genus Conus, and the fruit fly Drosophila (5-9). All chordates appear to contain the hemostatic VKD proteins (10). The known VKD proteins of Conus, however, have a distinct function where the VKD proteins are neurotoxic venom peptides (11)(12)(13)(14)(15). VKD proteins have not yet been isolated in Drosophila, and so the function of carboxylation in the fruit fly is not currently known.The carboxylase modifies VKD proteins by using O 2 and vitamin K hydroquinone (KH 2 ) to abstract the ␥-hydrogen of glutamyl residues to form a carbanion intermediate, which then incorporates CO 2 via nucleophilic attack to form the Gla (1, 2). During each Glu to Gla conversion, one molecule of KH 2 is oxidized to vitamin K epoxide, and the carboxylase is also an epoxidase. Insights into the molecular mechanism for this reaction have only recently been revealed. Early studies with thiol-specific inhibitors implicated Cys residues as part of the carboxylase active site (16). Chemical modeling based on those studies led to a proposed base strength amplification mechanism where a weak base (thiolate) initiates KH 2 oxygenation to generate a strong base that can abstract the

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Section: Free Vks Peptide Does Not Alter Carboxylase-vkd Protein Assomentioning

confidence: 99%

Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

Pudota

Hommema

Hallgren

et al. 2001

Journal of Biological Chemistry

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“…Carboxylase purifications have been attempted by using the propeptide as a ligand in affinity chromatography (15,16). The enzyme has also been isolated by virtue of its known association with its vitamin K-dependent protein precursors (13,20,21). A peptide representing human factor X propeptide was used to elute carboxylase activity from a-prothrombin (a-PT)-Sepharose (14).…”

Section: Introductionmentioning

confidence: 99%

“…Carboxylase activity was determined by incubating protein aliquots in a 228-,ul vol of 0.9 M ammonium sulfate/0.04% CHAPS/0.04% sodium cholate/0.04% phosphatidylcholine III-E/4 mM dithiothreitol/NaHl4CO3 (20 juCi; 400 nmol; Amersham)/0.9 mM Boc-Glu-Glu-Leu-OMe (EEL, Bachem). After addition of vitamin K hydroquinone (22) (5 tug), the samples were rocked at 200C, usually for 1 hr.…”

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confidence: 99%

Purification and identification of bovine liver gamma-carboxylase.

Berkner¹,

Harbeck²,

Lingenfelter³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The microsomal y-carboxylase catalyzes modification of a limited set of glutamyl residues to y-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes 02 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 105-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.The liver microsomal carboxylase catalyzes the posttranslational modification of selected glutamyl residues to Y-carboxylated glutamyl (Gla) residues in a limited set of proteins in a reaction requiring vitamin K hydroquinone, 02, and CO2 (1-3). These vitamin K-dependent proteins contain multiple glutamyl residues clustered at the N terminus in what is referred to as the Gla domain. Carboxylation of glutamyl residues in the Gla domain enables the Ca2+-mediated interaction ofthese proteins with phospholipids and is required for their biological activity. Vitamin K-dependent carboxylase activity has been detected in almost all mammalian tissues assayed and has been observed in one invertebrate as well (4). Its apparent presence in a wide variety of cultured lines can also be inferred from the ability of these lines to secrete carboxylated vitamin K-dependent recombinant proteins (5-10). A major limitation in understanding the mechanism of carboxylation has been the lack of a purified preparation of this enzyme. Attempts pmade using selective detergent extraction, ammonium sulfate fractionation, and conventional chromatography have been successful only in a limited purification (11-13). More recently, the propeptide sequence unique to all vitamin K-dependent proteins has been used in attempts to purify the carboxylase (14-16). This sequence is similar among vitamin K-dependent proteins that share very little other homology, leading to the proposal that the propeptide sequence is important for carboxylase recognition (17). The observations that mutations in protein C (18) or factor IX (19) impair carboxylation supported this concept. Carboxylase purifications have been attempted by using the propeptide as a ligand in affinity chromatography (15,16). The enzyme has also been isolated by virtue of its known association with its vitamin K-dependent prote...

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“…The PI of the precursor protein and the effect which carboxylation had on the p l were not determined. As isolated under in vitro conditions, a large fraction of the carboxylase enzyme appears to be complexed with its precursor protein substrates (DeMetz et al, 1981;Swanson & Suttie, 1982). Only a small fraction (approximately 25%) of the prothrombin precursor pool in microsomes from warfarin-treated rats acts as a substrate for the carboxylase in vitro, and the pool which acts as a substrate is that which is bound to the microsomal membrane in a complex with the carboxylase (Swanson & Suttie, 1982).…”

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Prothrombin biosynthesis: characterization of processing events in rat liver microsomes

Jc¹,

Suttie²

1985

Biochemistry

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Plasma and hepatic microsomal forms of rat prothrombin have been compared by sodium dodecyl sulfate-polyacrylamide electrophoresis and isoelectric focusing. The major prothrombin species that accumulated in the microsomes of rats treated with warfarin had a molecular weight of 78 500 and a pI in 8 M urea of 6.3-6.5. Plasma prothrombin had a molecular weight of 83 500 and a pI of 5.3-5.7. Microsomes from normal rat liver contain a second pool of precursor with a molecular weight of 83 500, and digestion with the glycosidase Endo H indicated that this form has been processed to contain complex carbohydrates, while the Mr 78 500 form is a high mannose form and is the substrate for the vitamin K dependent carboxylase. Treatment of rats with tunicamycin revealed that glycosylation was not essential for carboxylation or secretion from the liver. Comparison of the aglyco forms of prothrombin and its precursors suggests that the intracellular forms contain a basic, Mr approximately 1500 peptide that is missing from the plasma form of prothrombin.

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Vitamin K dependent in vitro production of prothrombin

Cited by 31 publications

References 30 publications

Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

Identification of Sequences within the γ-Carboxylase That Represent a Novel Contact Site with Vitamin K-dependent Proteins and That Are Required for Activity

Purification and identification of bovine liver gamma-carboxylase.

Prothrombin biosynthesis: characterization of processing events in rat liver microsomes

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