Partial purification of bovine liver vitamin K‐dependent carboxylase by immunospecific adsorption onto antifactor X

Metz, M. de; Vermeer, Cees; Soute, Berry A.M.; Scharrenburg, G. J. M. Van; Slotboom, Arend J.; Hemker, H.C.

doi:10.1016/0014-5793(81)80290-6

Cited by 58 publications

(36 citation statements)

References 13 publications

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Section: Methodsmentioning

confidence: 99%

“…De Metz et al (34) have found that liver microsomes from warfarin-treated cows accumulate uncarboxylated precursors of prothrombin, factor IX, and factor X. Although the prothrombin precursor would be expected to be present at higher concentrations, 69% of the carboxylase was complexed with factor X precursors, whereas 21% was associated with prothrombin and 8% with factor IX (34). Further evidence for the high affinity of the propeptide of factor X for the carboxylase is that warfarin treatment of HepG2 cells increases the level of factor X associated with the carboxylase with a concomitant decrease in the amount of prothrombin associated with the carboxylase (25).…”

Section: Methodsmentioning

confidence: 99%

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The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

Stanley¹,

Jin

Lin

et al. 1999

Journal of Biological Chemistry

112

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The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (K i ) toward a factor IX propeptide/ ␥-carboxyglutamic acid domain substrate. The K i values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are ϳ100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin Kdependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamates to ␥-carboxyglutamate (Gla) 1 in a number of proteins. Most vitamin K-dependent proteins are involved in the hemostatic process (prothrombin, factors VII, IX, and X, and proteins C, S, and Z), whereas two others (bone Gla protein and matrix Gla protein) are associated with bone (1-4). Two new putative vitamin K-dependent proteins of unassigned function, proline-rich Gla proteins (PRGP1 and PRGP2), were identified by sequence homology searches and are believed to be membrane proteins (5).A conserved eighteen-amino acid sequence essential for substrate recognition is found in all vitamin K-dependent proteins and was first identified by Pan and Price (6) based on sequence comparisons of the blood and bone vitamin K-dependent proteins. The conserved region is present as a propeptide sequence amino-terminal to the highly conserved Gla domains of the vitamin K-dependent blood proteins and is proteolytically removed to form the mature protein. With bone Gla protein, this sequence is also present as a propeptide amino-terminal to the mature form of the protein, whereas with matrix Gla protein the vitamin K-dependent propeptide-like sequence is part of the mature form of the protein (7). Confirmation of the importance of the propeptide sequence in carboxylation is demonstrated by experiments where deletion of the propeptide abrogates carboxylation of factor IX or protein C expressed in cell culture (8, 9). In addition, mutagenesis studies have identified a number of highly conserved amino acids (e.g. Phe Ϫ16, Ala Ϫ10, Leu Ϫ6) as well as les...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

Stanley¹,

Jin

Lin

et al. 1999

Journal of Biological Chemistry

112

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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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“…Although it has attracted considerable interest since its discovery (27), the protein has been refractory to complete purification because of problems with its solubilization and stability (27)(28)(29)(30)(31)(32)(33)(34)(35)(36). To obtain pure carboxylase for detailed mechanistic studies of the action of this enzyme, we have developed a purification based upon the affinity of the enzyme for a synthetic propeptide.…”

mentioning

confidence: 99%

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

Hubbard

Ulrich

Jacobs

et al. 1989

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

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The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form rcarboxygutam acid residues within the vitamin K-dependent proteins. This enzyme renizes the 'y-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood cogulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of igh concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25°C by reductive cleavage ofthe enzyme-propeptide complex in the presence ofdetergent and phospholipids. The eluted protein (Mr, 77,000)

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Partial purification of bovine liver vitamin K‐dependent carboxylase by immunospecific adsorption onto antifactor X

Cited by 58 publications

References 13 publications

The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

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

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

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