Vitamin K-dependent carboxylase: effect of detergent concentrations, vitamin K status, and added protein precursors on activity

Activity of the rat liver microsomal vitamin K-dependent carboxylase has been studied at various concentrations of detergent. The activity which could be solubilized by 0.25% Triton X-100 was low but could be greatly increased if vitamin K-deficient rats were given vitamin K a few minutes before they were killed. At higher concentrations of Triton, more activity was solubilized and this effect was not seen. In vitro carboxylation of endogenous microsomal proteins was decreased by 80-90% if vitamin K was administered 1 min before rats were killed, but the amount of assayable prothrombin precursor was decreased by only 20%. Decarboxylated vitamin K-dependent rat plasma proteins were not substrates for the carboxylase and did not influence peptide carboxylase activity significantly. Purified microsomal prothrombin precursors did, however, stimulate carboxylation of peptide substrate and were used as a substrate for the carboxylase in a preparation from precursor depleted vitamin K-deficient rats.     

Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase

The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-Leu-Glu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnCl2 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation's ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.     

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.     

Vitamin K-dependent carboxylase: Evidence for cofractionation of carboxylase and epoxidase activities,and for carboxylation of a high-molecular-weight microsomal protein

The vitamin K-dependent carboxylase from rat liver microsomes has been fractionated by submitting a crude preparation of this activity to chromatography on different column supports. A constant ratio of vitamin K epoxidation and vitamin K-dependent carboxylation was observed in all column fractions with good carboxylase activity, supporting the hypothesis that these two activities are carried out by the same enzyme complex. The preparation obtained (Complex B) is stable for several days when left on ice and has the same general properties as those observed in Triton X-100-solubilized microsomes. When antiserum raised against Complex B was incubated with Complex B, a twofold increase in carboxylase activity was observed. Benzidine staining showed that an appreciable pool of the antibody population was directed against hemeprotein(s). These data and spectral analyses indicated that a major contaminant of the preparation in cytochrome P-450. Although endogenous prothrombin precursors were absent in the crude starting preparation, a constant ratio of endogenous substrate carboxylation and carboxylation of a soluble substrate was observed during fractionation. A protein with a molecular weight of approximately 120,000 which copurified with Complex B was identified as substrate for the carboxylase.     

Vitamin K-dependent carboxylase: requirements for carboxylation of soluble peptide and substrate specificity.

Rat liver microsomes contain a triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of precursor proteins to γ-carboxyglutamyl residues. This activity has been studied utilizing synthetic peptides as substrates for the enzyme. When compared to the carboxylation of the endogenous microsomal precursors, the peptide carboxylase activity is more sensitive to the action of various inhibitors, and requires a higher concentration of vitamin K for maximal activity. The apparent K_m for the peptide Phe-Leu-Glu-Glu-Leu was found to be 4 mM. Substrate specificity depends on residues adjacent to the carboxylated Glu residues and macromolecular recognition sites.     

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.     

Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver

Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.     

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.     

Vitamin K dependent in vitro production of prothrombin

During prothrombin biosynthesis, glutamyl residues in prothrombin precursor proteins are carboxylated to gamma-carboxyglutamyl residues by a vitamin K dependent carboxylase. Calcium-dependent and calcium-independent rat prothrombin antibody subpopulations have been produced and utilized to study the liver microsomal precursors of prothrombin that accumulate when vitamin K action is blocked. A substantial portion of the precursor pool accumulating in the vitamin K deficient or warfarin-treated rat will react with a Ca2+-dependent antibody at high calcium concentration and appears to be partially carboxylated. During in vitro incubation in the presence of vitamin K, the fraction of the precursor pool which is tightly bound to the microsomal membrane appears to be the preferred substrate for the vitamin K dependent carboxylation. A small amount of completely carboxylated rather than a large amount of partially carboxylated products are produced during these incubations. Treatment with a Sepharose-bound prothrombin antibody demonstrated that about 20-25% of the total carboxylated microsomal protein precursor pool consists of prothrombin precursors. This treatment removes an equal amount of total carboxylase activity, and the enzyme is active in this carboxylase precursor-antibody complex.     

Synthesis of menaquinone-2 derivatives as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of a number of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) derivatives. The 2-des-methyl and 2-ethyl-MK-2 derivatives had very low activity as substrates. The 6- or 7-methyl-MK-2 derivatives and (6,7)-chloro-MK-2 were relatively high Vmax substrates with Km values increased over that seen for K-2. The 5- or 8-methyl-MK-2 derivatives were low Vmax substrates but also demonstrated low Km values. Although these observations suggested that 5-methyl-MK-2 might be a competitive inhibitor of the carboxylation reaction, it was not an effective inhibitor of either phylloquinone or 6-methyl-MK-2-dependent carboxylation.     

Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro

The vitamin K-dependent blood-clotting proteins contain a gamma-carboxylation recognition site in the propeptide, between the signal peptide and the mature protein, that directs gamma-carboxylation of specific glutamic acid residues. To develop a better substrate for the in vitro assay of the vitamin K-dependent gamma-carboxylase and to understand the substrate recognition requirements of the carboxylase, we prepared synthetic peptides based upon the structure of human proprothrombin. These peptides were employed as substrates for in vitro carboxylation using a partially purified form of the bovine liver carboxylase. A 28-residue peptide (HVFLAPQQARSLLQRVRRANTFLEEVRK), based on residues -18 to +10 in proprothrombin, includes the complete propeptide and the first 10 residues of acarboxyprothrombin. Carboxylation of this peptide is characterized by a Km of 3.6 microM. In contrast, FLEEL is carboxylated with a Km of about 2200 microM. A 10-residue peptide (ANTFLEEVRK), based on residues +1 to +10 in prothrombin, and a 20-residue peptide (ARSLLQRVRRANTFLEEVRK), based on residues -10 to +10 in proprothrombin, are also poor substrates for the carboxylase. Replacement of phenylalanine with alanine at residue 3 (equivalent to position -16 in proprothrombin) in the 28-residue peptide significantly alters the Km to 200 microM. A synthetic propeptide (HVFLAPQQARSLLQRVRRY), homologous to residues -18 to -1 in proprothrombin, inhibited carboxylation of the 28-residue peptide substrate with a Ki of 3.5 microM, but modestly stimulated the carboxylation of the 5- and 10-residue peptide substrates. These results indicate that an intact carboxylation recognition site is required for efficient in vitro carboxylation and that this site includes critical residues in region -18 to -11 of proprothrombin. The carboxylation recognition site in the propeptide binds directly to the carboxylase or to a closely associated protein.     

A comparison between vitamin K-dependent carboxylase from normal and warfarin-treated cows

Detergent-solubilized microsomal preparations that catalyse the vitamin K-dependent γ-carboxylation of glutamic acid residues in peptide and protein substrates, have been obtained from the livers of normal and warfarin-treated cows. The preparations from warfarin-treated animals contained more endogenous substrate than those from normal cows, but otherwise the two preparations were indistinguishable. The enzymes vitamin K reductase and γ-glutamyl carboxylase, may function independently of each other in this system. They are, nevertheless, intimately linked in some way, so that the reduced vitamin K that is produced by the former enzyme can be used immediately by the latter.     

Rat liver vitamin K-dependent carboxylase: a study of antibodies raised against partially purified preparations of the enzyme

Antibodies raised against three preparations of increasing purity of the microsomal vitamin K-dependent carboxylase did not neutralize essential proteins in the enzyme complex. When immobilized on Sepharose the antibodies removed 75% of contaminating proteins in the starting material, including cytochrome P-450. Immunoaffinity chromatography was more efficient when carried out in the presence of the detergent CHAPS than in the presence of Triton X-100. Immunoabsorption stimulated carboxylase activity 2.9-fold and resulted in a 66-fold increase in the specific activity of the complex.     

PROTEIN KINASES IN MYELIN OF RAT BRAIN: SOLUBILIZATION AND CHARACTERIZATION

Myclin from rat brain contained adenosine 3′, 5′-monophosphate (cyclic AMP)-dependent protein kinase activity, which was solubilized by 0.2% Triton X-100 and required exogenous protein substrate for its activity. Also present was a protein kinase which catalysed the phosphorylation of the endogenous substrate and which was neither solubilized by Triton X-100 nor stimulated by cyclic AMP. Sodium fluoride was required to maintain the activity of the endogenous phosphorylation, probably by inhibiting ATPase activity, but had no effect on the phosphorylation of histone by the solubilized enzyme. Protamine and myelin basic protein served as well as histone as a substrate for the solubilized enzyme. A protein kinase modulator had no effect on the endogenous phosphorylation, but inhibited histone phosphorylation by the solubilized enzyme. Cyclic AMP-binding activity was observed in both the solubilized and non-solubilized preparations. The concentration of cyclic AMP required to give half-maximal binding activity of the preparations was about 2.5 nM. The results indicate that the cyclic AMP-binding site of the protein kinase in myelin may partially be accessible, whereas the catalytic site may be integrated into the membrane structure of myelin.     

The stereochemistry of hydrogen abstraction in vitamin K-dependent carboxylation

The stereochemistry of the hydrogen abstraction in the vitamin K-dependent carboxylation of synthetic peptides has been investigated; the carboxylation rates of various peptidic substrates containing a stereospecifically 4-monodeuterated glutamic acid residue have been compared to that of nondeuterated peptides. A significant isotope effect was found only with the substrates containing (4S)-4-deuterated glutamic acid. These data reveal that the rat liver microsomal vitamin K-dependent carboxylase acts stereospecifically in abstracting the 4-pro-S hydrogen of the glutamyl residue. The low values of the measured isotope effects indicate that the hydrogen abstraction does not constitute a limiting step in the carboxylation mechanism.     

Vitamin K-dependent carboxylase. Stoichiometry of vitamin K epoxide formation, gamma-carboxyglutamyl formation, and gamma-glutamyl-3H cleavage

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of peptide-bound glutamyl residues to gamma-carboxyglutamyl (Gla) residues with the concomitant formation of vitamin K 2,3-epoxide (KO). These studies have demonstrated that the half-reaction, formation of KO, occurs in the absence of carboxylation at low glutamyl substrate concentration but that the ratio of KO/Gla approaches unity as the glutamyl substrate concentration is increased. Utilization of the carboxylase substrate Phe-Leu-[gamma-3H] Glu-Glu-Leu has demonstrated that the ratios of KO/gamma-C-H bonds cleaved and Gla/gamma-C-H bonds cleaved are equivalent at high substrate concentrations and that these ratios approach unity. At low substrate concentrations, KO formation occurs at a higher rate than gamma-H bond cleavage. These data are consistent with a mechanism involving the formation of an oxygenated intermediate from vitamin KH2 and O2 that is converted to KO during hydrogen abstraction from the gamma-position of the Glu substrate. In the absence of a Glu substrate, the intermediate is converted to KO by a mechanism not coupled to glutamyl activation.     

Bimodal frequency distribution of rat hepatic vitamin K-dependent carboxylation rate

The time course of vitamin K-dependent carboxylation was studied in an in vitro rat hepatic microsomal system. This method is based on incorporation of radiolabelled CO2 into endogenous substrate proteins. Forty rats were studied in order to characterize the intrinsic formation rate (V/KM) of carboxylated vitamin K-dependent proteins and the maximum amount of endogenous substrate available for vitamin K-dependent carboxylation (P infinity; normalized for the total amount of microsomal protein harvested). The frequency distributions of V/KM and P infinity values were both well described as the sum of two Gaussian components, each representing about 40% and 60% of the populations.     

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.     

Identification of a receptor for IgG on human granulocytes

The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected between these carboxylating systems with respect to their affinity for vitamin K and warfarin. Most of the endogenous substrates had some affinity towards antiprothrombin or antifactor X.     

Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.