The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats

The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.     

A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization.

Four proteins active in blood coagulation have long been known to require vitamin K for their proper biosynthesis: factors II, VII, IX, and X. This paper describes the purification of a hitherto unrecognized vitamin K-dependent glycoprotein from bovine plasma. The biosynthesis of this protein is interfered with by the vitamin K antagonist Dicoumarol. The molecular weight of the protein is approximately 56,000 and, like factor X, it has two polypeptide chains. The light chain binds Ca2+. Its NH2-terminal amino acid sequence is homologous to the NH2-terminal sequences of the other vitamin K-dependent proteins and it contains vitamin K-dependent gamma-carboxyglutamic acid residues. The biological function of this protein is unknown.     

Gas-6 and protein S: vitamin K-dependent factors and ligands for the TAM tyrosine kinase receptors family

The gamma-carboxyglutamate-containing proteins are a family of secreted vitamin K-dependent proteins in which some glutamyl residues are post-translationally modified to gamma-carboxyglutamic acid residues. A vitamin K-dependent gamma-glutamyl carboxylase enzyme catalyses this post-translational modification. The gamma-carboxylase reaction requires vitamin K in its reduced form, vitamin K hydroquinone, and generates gamma-carboxyglutamate and vitamin K 2,3,-epoxide which is then recycled back to the hydroquinone form by a vitamin K reductase system. Warfarin blocks the vitamin K cycle and hence inhibits the gamma-carboxylase reaction, and this property of Warfarin has led to its wide use in anticoagulant therapy. Until recently, interest in vitamin K-dependent proteins was mostly restricted to the field of hematology. However, the discovery that the anti-coagulant factor protein S and its structural homologue Gas6 (growth arrest-specific gene 6), two vitamin K-dependent proteins, are ligands for the Tyro3/Axl/Mer family of related tyrosine kinase receptors has opened up a new area of research. Moreover, the phenotypes associated with the invalidation of genes encoding vitamin K-dependent proteins or their receptors revealed their implication in regulating phagocytosis during many cell differentiation phenomena such as retinogenesis, neurogenesis, osteogenesis, and spermatogenesis. Additionally, protein S was identified as the major factor responsible for serum-stimulated phagocytosis of apoptotic cells. Therefore, the elucidation of the molecular mechanisms underlying the role of vitamin K-dependent proteins in regulating apoptotic cell phagocytosis may lead to a better understanding of the physiopathology of cell differentiation and could form the framework of new therapeutic strategies aiming at a selective targeting of cell phagocytosis associated pathologies.     

Vitamin K-dependent carboxylase: Possible artifact of analysis due to a pyridine nucleotide-dependent carboxylation

Addition of pyridine nucleotides to a microsomal system which is commonly used to study the vitamin K-dependent microsomal carboxylase promoted carboxylation of unknown endogenous compounds. Upon gel filtration, the carboxylated products were found to be of lower molecular weight (MW range 180–650) than the peptide substrate of the vitamin K-dependent carboxylase. Synthesis of these products was not inhibited by vitamin K antagonists nor did pyridine nucleotides stimulate carboxylation of the peptide substrate for vitamin K-dependent carboxylation in the absence of vitamin K. Thus the reaction appears to be mediated by a different enzyme. Dialysis of the microsomal system removed this pyridine nucleotide-stimulated carboxylation and activated the vitamin K-dependent carboxylation and epoxidation reactions. These data point out a possible artifact in the routine study of this enzyme and suggest that dialysis should be carried out prior to studying these two vitamin K-dependent reactions.     

Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.     

The identification of matrix Gla protein in cartilage

The vitamin K-dependent bone protein matrix gamma-carboxyglutamic acid (Gla) protein (MGP) has been identified by radioimmunoassay in the guanidine extract of rat cartilage. MGP was present in all cartilages tested at levels comparable to the MGP level in bone. Western blot analysis indicated that the molecular weight of cartilage MGP is the same as bone MGP, and Northern blot analysis revealed that MGP mRNA from cartilage is the same size as the MGP mRNA from bone. The structurally related vitamin K-dependent protein bone Gla protein could not be detected in cartilage by radioimmunoassay or by Northern blot analysis. The discovery that MGP is synthesized by growth plate cartilage could provide an explanation for the excessive growth plate mineralization disorder seen in rats treated with the vitamin K antagonist warfarin and the punctate mineralization of the growth plate seen in infants whose mothers received warfarin in the first trimester of pregnancy (the fetal warfarin syndrome). Both disorders appear to be caused by the inactivation of a vitamin K-dependent mineralization inhibitor in cartilage, an inhibitor which we suggest is MGP.     

Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.     

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10⁶). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor protein and increased, suggesting an analogous vitamin K mechanism as in liver.     

The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin.

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.     

Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.

During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.     

Engineering of a recombinant vitamin K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic acid forming capacity: evidence for a functional CXXC redox center in the system

The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.     

Glutamyl substrate-induced exposure of a free cysteine residue in the vitamin K-dependent gamma-glutamyl carboxylase is critical for vitamin K epoxidation.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamic acid to gamma-carboxyglutamic acid in the vitamin K-dependent proteins of blood and bone. The vitamin K-dependent carboxylase also catalyzes the epoxidation of vitamin K hydroquinone, an obligatory step in gamma-carboxylation. Using recombinant vitamin K-dependent carboxylase, purified in the absence of propeptide and glutamic acid-containing substrate using a FLAG epitope tag, the role of free cysteine residues in these reactions was examined. Incubation of the vitamin K-dependent carboxylase with the sulfhydryl-reactive reagent N-ethylmaleimide inhibited both the carboxylase and epoxidase activities of the enzyme. This inhibition was proportional to the incorporation of radiolabeled N-ethylmaleimide. Stoichiometric analyses using [(3)H]-N-ethylmaleimide indicated that the vitamin K-dependent carboxylase contains two or three free cysteine residues. Incubation with propeptide, glutamic acid-containing substrate, and vitamin K hydroquinone, alone or in combination, indicated that the binding of a glutamic acid-containing substrate to the carboxylase makes accessible a free cysteine residue that is important for interaction with vitamin K hydroquinone. This is consistent with our previous observation that binding of a glutamic acid-containing substrate activates vitamin K epoxidation and supports the hypothesis that binding of the carboxylatable substrate to the enzyme results in a conformational change which renders the enzyme catalytically competent.     

Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (greater than 90%) inhibited by l mM p-hydroxy-mercuribenzoate, and this inhibition is reversed by dithiothreitol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site vitamin K oxygenase contains an active thiol group.     

Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.     

Novel subunit in C4b-binding protein required for protein S binding

C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Absorption and metabolism of vitamin K in Swiss 3T3 mouse fibroblasts--a model system for study of vitamin K absorption and metabolism

The vitamin K cycle previously described in liver has been demonstrated in Swiss 3T3 mouse fibroblasts. Vitamin K epoxide and gamma-carboxyglutamic acid were isolated from the cells and chemically characterized. Menaquinone (MK4) is also metabolized to its epoxide and vitamin K epoxide is reduced to vitamin K in these cells. Thus Swiss 3T3 mouse fibroblasts provide a useful model system for the study of vitamin K metabolism. Possible functions of the vitamin K-dependent protein(s) in fibroblasts are discussed.     

Modification of the vitamin K-dependent carboxylase assay

Methods are presented that describe alternative protocols for the isolation of rat liver microsomes containing the vitamin K-dependent carboxylase and the procedure in which the solubilized enzyme is assayed. The method for determining the rate of 14CO2 incorporation into low molecular weight, acid soluble substrates by the rat liver microsomal vitamin K-dependent carboxylase has been modified in order to optimize safety, accuracy and simplicity. For these studies the rat liver microsomes containing the vitamin K-dependent carboxylase were isolated by CaCl2 precipitation. These Triton X-100 solubilized microsomes were found to be equivalent to the microsomes obtained by high speed ultracentrifugation with regard to protein concentration, pentapeptide carboxylase activity, carboxylase activity, preprothrombin concentration and total carboxylatable endogenous protein substrate. This modified assay procedure requires fewer steps and pipetting transfers and is quantitatively equivalent to previously employed protocols. The described technique can be adapted for any assay where 14CO2 or H14CO3- is incorporated into non-volatile products. This newly developed assay procedure was employed to assess conditions necessary for optimal vitamin K-dependent carboxylation of the less expensive substrate, N-t-Boc-L-glutamic acid alpha-benzyl ester. The optimal conditions for the carboxylation of N-t-Boc-L-glutamic acid alpha-benzyl ester by the carboxylase were found to be 10 mM N-t-Boc-L-glutamic acid alpha-benzyl ester, 10 mM MgCl2 at 15-18 degrees C. The rate of N-t-Boc-L-glutamic acid alpha-benzyl ester carboxylation under these optimized conditions was found to be higher (1.5-fold) than the rate of carboxylation of 1 mM Phe-Leu-Glu-Glu-Ile in the presence of the cation activator, MgCl2.     

Post-translational carboxylation of preprothrombin

Summary In summary, in this review on the function of vitamin K in post-translational modification of precursor proteins by carboxylation of certain glutamyl residues, I have tried to cover in particular the recent work on the reaction, the enzymes involved and the mechanisms being considered.In doing this I have also considered vitamin K, its discovery, its functional form and the possible relation of its metabolism to the carboxylation reaction. Equally the various vitamin K-dependent gla-containing proteins currently known have been described. The carboxylation of synthetic small molecule exogenous substrates and the synthesis and metabolism of the products of carboxylation are of great help in studying the reaction.Structural specificity of vitamin K analogs in vivo and in vitro has been compared and the use of various antagonists in vivo and in vitro considered in attempts to gain an understanding of the overall reaction.The reactions subsequent to carboxylation, e.g., the activation of prothrombin to thrombin via serine proteases and the related activation of the other vitamin K-dependent proteins have not been considered in this review. The review has not covered prothrombin or other vitamin K-dependent protein isolation, nor the determination of these proteins.As the vitamin K-dependent protein carboxylation story has developed over the past six years, a number of reviews have been written which help in keeping up with the various aspects of the field as it has expanded. These reviews refer to many of the papers I have had to eliminate due to space limitations. They are referenced as 469–489.The review is in no sense comprehensive and many papers have been missed or only mentioned. I have tried to concentrate on the more recent work and, thus, much of the very fine work of the 1940's on vitamin K chemistry is hardly mentioned.Some redundancy has been built into the organization of the review so that a reader can obtain a reasonable view of any one section without having to search the whole review for all possible relevant information on any particular part of the field.     

Cell-free translation of the vitamin K-dependent bone protein osteocalcin

The primary gene product of the vitamin K-dependent bone matrix protein, osteocalcin, has been identified by immunoprecipitation of cell-free translated proteins from 4 week rat calvariae mRNA preparations. Peptides of 9.8kd and 12kd, precipitated with a polyclonal affinity selected species specific antibody raised to purified rat osteocalcin, accounted for 1-2% of labelled proteins and were displaced by rat osteocalcin. These studies demonstrate that the 5800 molecular weight osteocalcin is synthesized as a precursor of approximately twice its size. The size of the propeptide, with a molecular weight of 4.3kd, is consistent with other known secreted vitamin K-dependent blood proteins.