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Biomedical subjects

C Vermeer

Publications and source records attributed to C Vermeer.

At least 73 records · Page 4Linked to original sources
PubMed1993

The relative effects of phylloquinone and menaquinone-4 on the blood coagulation factor synthesis in vitamin K-deficient rats.

Rats were made vitamin K-deficient by feeding them a 1:1 (w/w) mixture of a commercial vitamin K-depleted diet and boiled white rice. After one week of treatment the rats had developed severe vitamin K deficiency, resulting in Thrombotest values of 5-10% of the initial values. In this experimental system the efficacy of phylloquinone (K1) was compared with that of menaquinone-4 (MK-4) by measuring the extent to which the Thrombotest was normalized after the administration of varying doses of the respective vitamins. Oral administration of the vitamins showed that the efficacy of K1 was at least two-fold higher than that of MK-4. As comparable results were obtained after subcutaneous administration of the vitamins, we conclude that after oral administration the intestinal absorption had been quick and nearly complete. A less pronounced effect of K1 and MK-4 was found after colorectal administration. For both forms of vitamin K relatively high amounts (well above the physiological concentration) were required before significant effects on the Thrombotest could be observed. Therefore these data demonstrate the importance of sufficient dietary vitamin K consumption in rats. The efficacy of other menaquinones may be investigated in the same experimental animal model system.

Administration, Oral↗
PubMed1993

Characterization of the purified vitamin K-dependent gamma-glutamyl carboxylase.

Vitamin K-dependent carboxylase, purified from bovine liver, has properties similar to those reported for the carboxylase activity present in crude, solubilized microsomes. The purified carboxylase was found to possess the vitamin K epoxidase activity, believed to be essential for vitamin K-dependent carboxylation, but did not contain vitamin K epoxide reductase activity. Kinetic studies of the carboxylase done under defined conditions were complicated by the non-Michaelis-Menten kinetic behavior observed for reactions with two of the enzymes substrates, FLEEL and vitamin K1 hydroquinone. Initial rate experiments with the substrate FLEEL demonstrated behavior consistent with substrate inhibition and gave half-maximal activity at 1 mM FLEEL. Experiments with the substrate vitamin K1 hydroquinone also displayed non-Michaelis-Menten kinetics, as maximal activity was reached prematurely in relation to behavior at lower concentrations. Half-maximal activity was observed at 35 microM vitamin K1 hydroquinone. Initial rate experiments with varying NaH14CO3 concentration displayed Michaelis-Menten kinetics and gave a Km(app) of 0.29 mM. At cosubstrate concentrations chosen to obtain near-maximal activity, initial rate studies with varying NaH14CO3 concentration indicated a kcat near 1.0 s-1. Removal of the fourth substrate, oxygen, resulted in the loss of more than 99% of carboxylase activity. The sulfhydryl reagent N-ethylmaleimide inhibited carboxylase irreversibly, as did the anticoagulant warfarin.

Amino Acid Sequence↗
PubMed1993

Vitamin K-induced changes in markers for osteoblast activity and urinary calcium loss.

The objective of this study was to identify subjects in whom vitamin K has an effect on markers for calcium and bone metabolism and to detect hitherto-unnoticed correlations between vitamin K-induced changes in these markers. Participants in our studies were apparently healthy women, in whom we measured serum-immunoreactive osteocalcin (irOC) before and after adsorption to hydroxylapatite; total serum alkaline phosphatase (T-AP) and bone-specific alkaline phosphatase (B-AP); and fasting urinary calcium and creatinine. We describe a trial among 145 women who were treated with vitamin K (1 mg/day) for 2 weeks, and a prospective placebo-controlled trial among two groups each of 70 postmenopausal women with a treatment period of 3 months. It turned out that in elderly women vitamin K induced increased levels of serum irOC with a high affinity for hydroxylapatite (irOCbound), whereas that with low affinity (irOCfree) remained unaffected. In placebo-treated women the ratio irOCfree/irOCbound shifted from 0.38 to 0.65 around the 50th year of age. This shift was not found in vitamin K-treated women. After 3 months of treatment the vitamin K-induced changes in irOCbound were correlated with changes in B-AP, whereas irOCfree was correlated to urinary calcium excretion. In fast losers of urinary calcium vitamin K induced a 30% decrease of calcium excretion. The hypothesis is put forward that irOCbound may be a marker for bone formation, that serum irOCfree may be a marker for bone resorption, and that the serum irOCfree/irOCbound ratio may become a marker for skeletal remodeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Effects of vitamin K and oral anticoagulants on urinary calcium excretion.

In a subgroup of postmenopausal women vitamin K induced a decrease of the urinary calcium loss. This effect was significant (P < 0.0001) in the so-called fast losers of calcium (calcium/creatinine ratio > 0.5). To find out whether vitamin K antagonists would have an opposite effect, a study was started among 141 persons on long-term oral anticoagulant therapy. In this population the number of fast losers was recorded, and compared to that in a group of age- and sex-matched non-treated controls. Notably in young men the fraction of fast losers was significantly higher in the anticoagulant-treated group than in the control group (25 v 0%, P < 0.02). Differences between treated and nontreated groups may also be found in other markers for calcium and bone metabolism, notably in serum osteocalcin concentration and in urinary hydroxyproline excretion. The conclusion of our study is that oral anticoagulant treatment must be regarded as a potential risk factor for a high loss of urinary calcium.

Adult↗
PubMed1992

Congenital deficiency of all vitamin K-dependent blood coagulation factors due to a defective vitamin K-dependent carboxylase in Devon Rex cats.

Two Devon Rex cats from the same litter, which had no evidence of liver disease, malabsorption of vitamin K or chronic ingestion of coumarin derivatives, were found to have plasma deficiencies of factors II, VII, IX and X. Oral treatment with vitamin K1 resulted in the normalization of these coagulation factors. After taking liver biopsies it was demonstrated that the coagulation abnormality was accompanied by a defective gamma-glutamyl-carboxylase, which had a decreased affinity for both vitamin K hydroquinone and propeptide. This observation prompted us to study in a well-defined in vitro system the possible allosteric interaction between the propeptide binding site and the vitamin K hydroquinone binding site on carboxylase. It was shown that by the binding of a propeptide-containing substrate to gamma-glutamylcarboxylase the apparent KM for vitamin K hydroquinone is decreased about 20-fold. On the basis of these in vitro data the observed defect in the Devon Rex cats can be fully explained.

Amino Acid Sequence↗
PubMed1992

Serum osteocalcin as a marker for vitamin K-status in pregnant women and their newborn babies.

Osteocalcin (bone Gla-protein) is a vitamin K-dependent protein synthesized by osteoblasts. Its hydroxylapatite binding capacity (HBC) is generally used to estimate the Gla-content of circulating osteocalcin. Here we have used the HBC of serum osteocalcin as a marker for the vitamin K-status in pregnant women and their offspring. For all cases investigated the HBC values in the cord samples were substantially lower than in the corresponding maternal ones. Babies from mothers who had been treated with vitamin K during the last 6 weeks prior to delivery, had significantly higher HBC values than those from a placebo group. The results presented in this paper are indicative for a generally occurring vitamin K deficiency in newborns. At delivery the HBC in untreated women was low as well. In both the placebo- and the vitamin K-group a good correlation was found between the HBC values in paired samples from mother and child. Whether the maternal HBC value may be used as a prenatal marker for estimating the fetal vitamin K-status remains to be seen.

Adsorption↗
PubMed1992

Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system. Strong synergistic effects with protein disulphide-isomerase.

It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes. Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above. Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor. In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher. PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM. These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues. In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells. The possibility that the reactions are also coupled in vivo is discussed.

Animals↗
PubMed1991

Vitamin K-dependent carboxylation of poly-L-glutamate.

Poly-L-glutamate preparations of varying chain length were used as substrates for bovine liver vitamin K-dependent carboxylase. The quality of these substrates (as measured by their apparent kinetic constants) was comparable to that of the more commonly used tri- and pentapeptides, but the maximal reaction rate increased at increasing chain length.

Animals↗
PubMed1990

In vitro gamma-carboxylation of a 59-residue recombinant peptide including the propeptide and the gamma-carboxyglutamic acid domain of coagulation factor IX. Effect of mutations near the propeptide cleavage site.

We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla). CNBr was used to release FIXGla from the fusion protein. The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation. Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B. R., Jacobs, M., Ulrich, M. M. W., Walsh, C., Furie, B., and Furie, B. C. (1989) J. Biol. Chem. 264, 14145-14150). In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule). We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation. Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates. Maximum carbon dioxide incorporation was achieved with the double mutant. From these data we conclude the following. 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase. 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.

1-Carboxyglutamic Acid↗
PubMed1990

Comparison of the vitamins K1, K2 and K3 as cofactors for the hepatic vitamin K-dependent carboxylase.

The vitamins phylloquinone (K1), menadione (K3) and various menaquinones (K2) were compared for their ability to serve as a cofactor for the hepatic vitamin K-dependent carboxylase. It was found that the cofactor activity of the menaquinones varied with the length of the aliphatic side-chain and showed an optimum at MK-3. Menadione was not active at all. The concentration required for half-maximal reaction velocity (K 1/2) was determined for the various menaquinones and decreased at increasing chain length. The K 1/2 value for MK-4 was 3-times lower than that for vitamin K1. Under our in vitro conditions both vitamin K1 and the K2 vitamins were rapidly metabolized into a mixture of the quinone, the hydroquinone and the epoxide form. The fact that at equilibrium the level of these three metabolites was independent of the starting material shows that the vitamin K cycle is operational for vitamin K1 as well as for K2.

Amino Acid Sequence↗
PubMed1990

Vitamin K-dependent carboxylase: effect of ammonium sulfate on substrate carboxylation and on inhibition by stereospecific substrate analogs.

(1) High concentrations of ammonium sulfate may stimulate the carboxylase activity of bovine liver microsomes about 10-fold. This effect results from an increase of the Vmax, whereas neither the apparent Km for a number of substrates nor the Ki for substrate analogs is affected. (2) The effect of ammonium sulfate was only found in substrates lacking the pro-sequence. No effect was measurable on the carboxylation of pro-PT28 and endogenous precursor proteins. (3) If the pro-fragment was added as a peptide not covalently bound to a carboxylatable substrate, the carboxylation thereof was only slightly affected and ammonium sulfate remained active as a stimulator of carboxylase activity. (4) S-MeTPT is a much stronger inhibitor of carboxylase activity than is R-MeTPT. (5) The inhibition of carboxylase by the methylated tripeptides is competitive and independent of the type of substrate. Also pro-PT28, which contains the full pro-sequence, could be inhibited completely. (6) On the other hand the carboxylation of endogenous protein precursors could only be partly inhibited by the substrate analogs: even at high concentrations of S-MeTPT a residual endogenous substrate carboxylation of about 30% was left.

Ammonium Sulfate↗
PubMed1989

The effect of vitamin K supplementation on circulating osteocalcin (bone Gla protein) and urinary calcium excretion.

STUDY OBJECTIVE: To determine whether vitamin K administration affects urinary calcium excretion in postmenopausal women. DESIGN: Before- and after-trials with a 2-week treatment period. SUBJECTS: Healthy postmenopausal women (55 to 75 years old) were recruited from the convents in and around Maastricht. Controls (25 to 40 years old) were healthy premenopausal volunteers. INTERVENTION: Daily administration of 1 mg of vitamin K for 2 weeks. MEASUREMENTS: Serum immunoreactive osteocalcin: hydroxylapatite binding (HAB) capacity of serum immunoreactive osteocalcin; excretion of calcium, hydroxyproline, and creatinine in the urine during the last 2 h of a 16-h fasting period. RESULTS: In premenopausal women, no effect of vitamin K administration was seen. In the postmenopausal group, vitamin K induced increased serum immunoreactive osteocalcin concentration; normalization of the HAB capacity of serum immunoreactive osteocalcin (this marker was less than 50% that of the controls in the pretreatment samples); a decrease in urinary calcium excretion, notably in the "fast losers" of calcium; and a parallel decrease in urinary hydroxyproline excretion in the fast losers of calcium. CONCLUSIONS: The serum immunoreactive osteocalcin level may vary with vitamin K status. This variance should be taken into consideration if osteocalcin is used as a marker for osteoblast activity. Vitamin K is one factor that may play a role in the loss of bone mass in postmenopausal osteoporosis.

Adult↗
PubMed1989

Vitamin K-dependent carboxylase: minimized escape of CO2 from solution may prolong linearity of the reaction rate.

Escape of 14CO2 from the reaction mixture into the gas phase may seriously affect the accuracy of in vitro measurement of vitamin K-dependent carboxylase activity (and probably that of other carboxylases as well). In this paper we describe the effect of (a) the volume of the test tubes in which the reaction is performed, (b) the addition of an excess of NaH12CO3 in parallel with standard amounts of NaH14CO3, and (c) the incubation temperature. In this way optimal conditions are defined and used for the carboxylation of various peptide and protein substrates. It is shown that both a prosequence and an internal recognition site contribute to the effective recognition of a substrate by carboxylase. The maximal efficiency of carboxylation was 1-2% with substrates lacking both signals and 20-50% if only one was present. This indicates the need for developing peptide substrates containing both recognition signals for vitamin K-dependent carboxylase.

Animals↗
PubMed1989

In vitro carboxylation of a blood coagulation factor IX precursor produced by recombinant-DNA technology.

Blood coagulation factor IX (Christmas factor) is a plasma protein which is required for normal haemostasis. A functional deficiency of factor IX results in haemophilia B, a bleeding disorder which is generally treated by infusions of factor IX concentrates prepared from pooled human plasma. The use of human blood products is connected with the risk of transmitting viral agents responsible for diseases such as hepatitis B and AIDS. Recombinant DNA techniques may provide the means to produce the required proteins without exposing the patients to these risks and at lower costs. One of the problems which has to be overcome before recombinant factor IX can be used for therapeutical purposes is related to the vitamin K-dependent carboxylation of its 12 NH2-terminal glutamate residues. In cell cultures this carboxylation, which is required to render the protein its procoagulant activity, is far from complete, especially at high expression levels. In this paper we describe the in vitro carboxylation of non and/or partly carboxylated recombinant factor IX produced by transformed Chinese hamster ovary cells. The identity of the newly formed Gla residues was verified and it could be demonstrated that all carboxyl groups had been incorporated into the recombinant factor IX.

Animals↗
PubMed1989

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

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation 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 high 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 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme.

Amino Acid Sequence↗