Vitamin K-dependent Carboxylation System Research Articles

Dexamethasone Stimulates Vitamin K-Dependent Carboxylase Activity in Neonatal Rats and Cultured Fetal Hepatocytes

gamma-Carboxylation of vitamin K-dependent clotting factors may be a key regulatory element in output of these proteins from liver to blood in the developing neonate. We have investigated the effect of hormones and growth factor on the vitamin K-dependent carboxylation system in neonatal rats and cultured fetal hepatocytes. Of the hormones and growth factor tested, only dexamethasone had a significant effect on the system. When dexamethasone was administered to newborn rats, there was a delayed response that produced significant enhancement of carboxylase activity 6 d after injection of the drug. A similar delayed response to the drug could also be demonstrated in cultured fetal hepatocytes. When cultured in the presence of 0.1-1 microM dexamethasone, cellular carboxylase activity was little affected by the drug the first 2 d of culture but the activity was increased more than threefold by 4 d in culture. Microsomal membranes from neonatal rat livers and fetal hepatocytes treated with dexamethasone showed enhanced vitamin K-dependent 14C labeling of the factor X membrane precursor pool. Enhanced labeling of the factor X membrane precursor pool has also been demonstrated in rats and HepG2 cells treated with warfarin. The data suggest that 1) gamma-carboxylation of clotting factors is regulated by glucocorticoids in the developing liver, and 2) dexamethasone stimulates intracellular gamma-carboxylation of the factor X precursor by a mechanism that currently is unknown.

The effects of warfarin on HepG2 cells suggest that prothrombin and factor X interact differently with the vitamin K-dependent carboxylase in the secretory pathway

HepG2 cells have been shown to respond to warfarin by 1) enhanced vitamin K-dependent carboxylase activity; 2) enhanced intracellular concentration of the factor X clotting factor precursor and 3) enhanced vitamin K-dependent 14C-labelling of a 74 kDa microsomal protein which has been identified as the factor X precursor. There was no difference in any of these measured parameters whether the cells had been treated for 4 or 24 hours with warfarin. In contrast to the intracellular factor X concentration, the intracellular prothrombin precursor concentration was not affected by the drug which suggests there is a difference in the mechanism of processing of these two clotting factors by HepG2 cells. The data are consistent with the view that warfarin maintains its effect on the vitamin K-dependent carboxylation system in HepG2 cells for 24 hours and support the hypothesis that clotting factor X and prothrombin precursors interact differently with the vitamin K-dependent carboxylase.

Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 1.6.99.2).

A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4-hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol-sensitive NAD(P)H:quinone reductase (EC 1.6.99.2; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4-hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system.

Vitamin K-Dependent Carboxylation in Articular Chondrocytes

A nonhepatic vitamin K-dependent protein, matrix Gla protein, has recently been identified in cartilage where it may play an important role in the control of mineralization or matrix development. We have investigated the vitamin K cycle in chondrocytes isolated from bovine and rabbit articular cartilage and examined these cells for their ability to synthesize vitamin K-dependent proteins. Chondrocytes were found to have an active vitamin K-dependent carboxylation system. Preincubation of the cells with warfarin resulted in a significant increase in the measured carboxylase activity. Both vitamin K epoxide reductase and DT-diaphorase (EC 1.6.99.2) activity were present indicating that chondrocytes are capable of producing reduced vitamin K1H2, the cofactor for the vitamin K-dependent carboxylase. Specific 14C-labeling of microsomal vitamin K-dependent protein precursors demonstrated synthesis of several vitamin K-dependent proteins by chondrocytes. 35S-labeling of chondrocyte proteins provided evidence that matrix Gla protein is synthesized by these cells.

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 Carboxylation in the Developing Rat: Evidence for a Similar Mechanism of Action of Warfarin in Fetal and Adult Livers

Our report presents data on maturation of the vitamin K-dependent carboxylation system in fetal and neonatal rat livers. This system which converts precursors of clotting factors II, VII, IX, X, protein S, and protein C to gamma-carboxylated proteins exhibited low gamma-carboxylation activity before birth. However, around the time of birth there was a sudden increase in all enzyme activities associated with the vitamin K-dependent carboxylation system. In 2-d neonatal rats these activities dropped to levels that were measured in fetal livers whereupon the activities had risen to adult levels in 7-d neonatal rats. However, the activities of the two pathways that provide the carboxylase with reduced vitamin KH2 cofactor were never as high as that measured in maternal livers. It appeared that the pathway which is insensitive to coumarin anticoagulant drugs matures later than the coumarin drug-sensitive pathway. This conclusion is supported by the finding of a late appearance in development of the vitamin K-reducing enzyme DT-diaphorase. Warfarin, when administered to the mother, affected the fetal livers at all stages of development studied (d 16-21). This was clearly demonstrated by vitamin K-dependent 14C-labeling of a 70-kD liver protein that has been shown previously to be a marker for the effect of this drug on the liver. The data demonstrate a similar mechanism of action of warfarin in fetal and neonatal rat livers and an ongoing maturation process of the vitamin K-dependent carboxylation system in these rats.

Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme.

The antidotal effect of vitamin K in overcoming poisoning by coumarin anticoagulant drugs is mediated by a vitamin K-reducing enzyme of the endoplasmic reticulum [Wallin & Martin (1987) Biochem. J. 241, 389-396]. With microsomes obtained from human liver biopsies, we have investigated the localization and the transverse orientation of this enzyme in the endoplasmic reticulum and compared its orientation to that of the other enzymes of the vitamin K-dependent carboxylation system. All enzymes were protected by the microsomal membrane and thus appear to have a luminal orientation in the endoplasmic reticulum, consistent with their role in the vitamin K-dependent modification of secretory glycoproteins. Separation of rough and smooth microsomes showed that vitamin K-dependent carboxylase activity was 6-fold higher in rough than in smooth microsomes. Vitamin K1 reduction by the coumarin-drug-sensitive (pathway I) and -insensitive (pathway II) enzymes of the vitamin K-dependent carboxylation system was the same in rough and smooth microsomes. The data suggest a close association between the pathway I and II enzymes in the endoplasmic reticulum. These pathways may be partial reactions of multienzyme complex which carries out the various activities associated with the vitamin K-dependent carboxylation system.

3-methylcholanthrene induction of enzymes in the vitamin K-dependent carboxylation system

The effect of 3-methylcholanthrene on liver enzymes in the vitamin K-dependent carboxylation system has been investigated in normal rats and rats treated with the anticoagulant warfarin. It was found that 3-methylcholanthrene did not interfere with the anticoagulant function of the drug. Treatment of rats with 3-methylcholanthrene resulted in a 2.7-fold increase in liver cytosolic DT-diaphorase activity and a 1.5-fold increase in liver microsomal vitamin K-dependent carboxylase activity. A pathway for production of reduced vitamin K cofactor for the vitamin K-dependent carboxylase is catalyzed by DT-diaphorase and an as yet unidentified NADH-specific dehydrogenase(s). The data suggest that the unidentified enzyme(s) in the pathway is not induced by 3-methylcholanthrene.

Effects of 1,25-dihydroxycholecalciferol administration on the rat renal vitamin K-dependent carboxylating system

We have shown previously that the in vitro activity of the renal vitamin K-dependent γ-glutamyi carboxylase toward synthetic oligopeptide substrates is stimulated by administration of either parathyroid hormone (PTH) or 1,25-dihydroxycholecalciferol [1,25 (OH) 2D 3] to rats [(1983) J. Biol. Chem. 258, 12783-12786]. Here we report that administration of 1,25(OH) 2D 3 to rats increases their levels of endogenous carboxylase substrate as well. Rats fed a vitamin D-deficient diet had highly elevated serum PTH levels while vitamin D-replete animals had undetectable levels. Furthermore, since PTH increases 1,25(OH) 2D 3 levels by stimulating renal 25-hydroxy vitamin D-1α-hydroxylase, it is very likely that the stimulatory effects of PTH on the renal vitamin K-dependent carboxylating system are mediated by 1,25(OH);,D3.

Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin.

The systems involved in vitamin K-dependent carboxylation and vitamin K metabolism have been extensively studied in rat liver. To determine how clinically applicable this information is, similar in vitro studies were completed using human liver. One major difference exists in the pathways that provide reduced vitamin K1 cofactor for the carboxylation reaction. The coumarin-sensitive DT-diaphorase (EC.1.6.99.2) in human liver appears to play a relatively minor role in the dehydrogenase pathway. However, similar to rat liver, the human liver contains a warfarin-insensitive enzyme in this dehydrogenase pathway. The data suggest that this enzyme is responsible for the antidotic effect of vitamin K1 in cases of coumarin intoxication. Human vitamin K epoxide reductase, which constitutes the other pathway for vitamin K1 reduction, has kinetic and enzymological characteristics that are very similar to the rat enzyme. This enzyme exhibited similar activity in rat and human microsomes. Initial velocities for vitamin K1 epoxide reduction in rat and human microsomes were 20 and 32 pmol/mg X min, respectively. The human enzyme is highly sensitive to warfarin inhibition. The mechanism for this inhibition appears to be similar to what has been proposed for the rat enzyme. Also, a vitamin K-dependent carboxylation system is described that allows both pathways to support the carboxylation reaction with reduced vitamin K1 cofactor. The effect of warfarin on this in vitro system is consistent with the current model for the mechanism of action of coumarin anticoagulant drugs in the rat.

Effects of parathyroid hormone and vitamin D on the renal vitamin K-dependent carboxylating system.

The vitamin K-dependent production of gamma-carboxyglutamic acid-containing protein occurs in the kidney. This protein, which we will call KGP, has no assigned physiological role. In the experiments described here, the effects of the calcium-regulating hormones, parathyroid hormone and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), on the renal vitamin K-dependent carboxylation system were studied. PTH, when administered via continuous infusion at 3 units/h using a subcutaneous osmotic pump, caused a time-dependent increase in the activity of the renal gamma-glutamyl carboxylase which was detectable after 2 days and continued to increase up to day 7 of infusion. The increase was more marked in thyroparathyroidectomized rats than in intact rats, suggesting the possibility of an antagonistic action by endogenous calcitonin. Treatment of intact rats with 1,25-(OH)2D3 by daily intraperitoneal injections also caused a time-dependent increase in the activity of the carboxylase. When the warfarin-mediated accrual in renal microsomes of the precursor of KGP was measured, treatment with either parathyroid hormone plus warfarin or 1,25-(OH)2D3 plus warfarin caused a significant increase in precursor relative to animals treated with warfarin alone. Thus, the synthesis of KGP is modulated by these two calcium regulating hormones both by increasing the synthesis of KGP precursor as well as by increasing the activity of the carboxylase enzyme which catalyzes post-translational modification of the precursor.

A new detergent for the solubilization of the vitamin K-dependent carboxylation system from liver microsomes: Comparison with triton X-100

A new nondenaturing zwitterion detergent has been used to solubilize the vitamin K-dependent carboxylating system from rat liver microsomes. The optimal conditions for solubilization were 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 1 m NaCl while maximum carboxylating activity was achieved with 0.5% CHAPS, 0.75 m NaCl for a microsomal protein concentration varying from 7.5 to 10 mg/ml. Using CHAPS, high levels of carboxylase activity can be found not only in liver microsomes from vitamin K-deficient rats, but also in microsomes prepared from the livers of normal animals, normal animals supplemented with vitamin K 1, or vitamin K-deficient animals supplemented with vitamin K 1, while little if any activity is detected after Triton X-100 solubilization of the microsomes of such livers. Thus, the new zwitterion detergent proves to be more effective and less denaturing in solubilizing the vitamin K-dependent carboxylase from rat liver microsomes than other detergents used in this system.

Fractionation and reconstitution of vitamin K-dependent carboxylation activity in rat liver microsomes.

Attempts to purify the vitamin K-dependent carboxylation system from rat liver microsomes have not yet met with success. Purification procedures result in low yields of activity even after relatively mild separation methods. We have been able to demonstrate that one reason for these failures is that there are at least two components (which can be separated) necessary for the reaction. Recombination of the separated components is possible if the detergent (Triton X-100) concentration is lowered and ethylene glycol is added to the system. Under these conditions, vitamin K-dependent carboxylation of synthetic pentapeptide (Phe-Leu-Glu-Glu-Leu) can be totally reconstituted.

Characteristics of the vitamin K-dependent carboxylating system in human placenta

Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin), VII, IX and X [1–4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO 2, O 2 and the reduced (hydroquinone) from of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K 1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12–14]. In on of these [14], a survey of rat tissues for vitamin K 1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.

Studies on a Subcellular System for Vitamin K- Dependent Carboxylation

The subcellular localization of acarboxyprothrombin and the vitamin K-dependent carboxylation system has been studied using livers from vitamin K-deficient rats and found to be mainly in the membranes of the rough microsomal fraction. Bovine acarboxyprothrombin or fragment 1 thereof were inactive as substrates in the rat carboxylating system.

The vitamin K-dependent synthesis of gamma-carboxyglutamic acid by bone microsomes.

Microsomes prepared from embryonic chick bone contain a vitamin K-dependent carboxylating system which post-translationally converts glutamic acid residues in peptides to gamma-carboxyglutamic acid (gamma-CGlu). Glutamic acid residues in both endogenous chick bone microsomal protein and in the synthetic peptide Phe Leu-Glu-Glu-Val are gamma-carboxylated. These data suggest that bone cells have the capacity for de novo gamma-CGlu synthesis and may be responsible for synthesis of osteocalcin, the major gamma-CGlu protein in bone.

New developments in the study of the vitamin K-dependent carboxylation system.

New developments in the study of the vitamin K-dependent carboxylation system.

Soluble enzyme system for vitamin K-dependent carboxylation.

The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.

Some characteristics of a vitamin K-dependent carboxylating system from rat liver microsomes

Vitamin K-dependent carboxylation of protein has been obtained in microsomes from K-deficient rats by supplementing the microsomes with either a reduced pyridine nucleotide or dithiothreitol. γ-Carboxyglutamate residues have been directly identified as the radioactive reaction product after incubating the microsomes with NaH 14CO 3 and vitamin K. Evidence is presented that vitamin K is reduced to the hydroquinone prior to its involvement in carboxylation and that dithiothreitol has an additional role of protecting a critical sulfhydryl group. A structure-activity study using a variety of vitamins K and related compounds is reported.