scholarly journals Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents

1994 ◽  
Vol 297 (2) ◽  
pp. 277-280 ◽  
Author(s):  
H H W Thijssen ◽  
Y P G Janssen ◽  
L T M Vervoort
Keyword(s):  
Vitamin K ◽  
Lipoic Acid ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Vitamin K Cycle ◽  
Redox Active ◽  
Starting Point ◽  
Epoxide Reductase ◽  
Triton X ◽  
Reducing Equivalents

This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.

scholarly journals Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

2022 ◽  
Author(s):  
Thomas Chetot ◽  
Etienne Benoit ◽  
Véronique Lambert ◽  
Virginie Lattard
Keyword(s):  
Vitamin K ◽  
Protein Disulfide Isomerase ◽  
Reductase Activity ◽  
Functional Characterization ◽  
Hek293 Cells ◽  
Vitamin K Epoxide Reductase ◽  
Disulfide Isomerase ◽  
Vitamin K Cycle ◽  
Epoxide Reductase ◽  
Protein Disulfide

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

Effect of warfarin on plasma and liver vitamin K levels and vitamin K epoxide reductase activity in relation to plasma clotting factor levels in rats

1990 ◽  
Vol 57 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Yoshitaka Yamanaka ◽  
Masahiro Yamano ◽  
Kojiro Yasunaga ◽  
Tsutomu Shike ◽  
Kiyohisa Uchida
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Clotting Factor ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase ◽  
Liver Vitamin

Vitamin K epoxide reductase activity in the metabolism of epoxides

1985 ◽  
Vol 34 (15) ◽  
pp. 2617-2620 ◽  
Author(s):  
I. Liptay-Reuter ◽  
K. Dose ◽  
T. Guenthner ◽  
W. Wörner ◽  
F. Oesch
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase

Purification of a Single Peptide with Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH2 Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 331-331
Author(s):  
Pei-hsuan Chu ◽  
Teng-yi Huang ◽  
Jason Williams ◽  
Darrel W. Stafford
Keyword(s):  
Vitamin K ◽  
Complete Recovery ◽  
Vitamin K Epoxide Reductase ◽  
Vitamin K Cycle ◽  
The Us ◽  
Epoxide Reductase ◽  
Measurable Activity ◽  
Turn Over ◽  
Single Peptide

Abstract More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy

1989 ◽  
Vol 38 (16) ◽  
pp. 2693-2701 ◽  
Author(s):  
Takashi Matsubara ◽  
Akira Touchi ◽  
Toshio Harauchi ◽  
Kyoji Takano ◽  
Toshio Yoshizaki
Keyword(s):  
Vitamin K ◽  
Reductase Activity ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase

Pharmacodynamic resistance to warfarin associated with a Val66Met substitution in vitamin K epoxide reductase complex subunit 1

2005 ◽  
Vol 93 (01) ◽  
pp. 23-26 ◽  
Author(s):  
Dominic Harrington ◽  
Sarah Underwood ◽  
Colin Morse ◽  
Martin Shearer ◽  
Edward Tuddenham ◽  
...  
Keyword(s):  
Vitamin K ◽  
Dose Response ◽  
Reductase Activity ◽  
Warfarin Dose ◽  
Coagulation Factor ◽  
Gene Encoding ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase ◽  
Undetectable Serum ◽  
Warfarin Resistance

SummaryThe gene encoding vitamin K epoxide reductase complex subunit 1 (VKORC1), a component of the enzyme that is the therapeutic target site for warfarin, has recently been identified. In order to investigate the relationship betweenVKORC1 and warfarin dose response, we studied theVKORC1 gene (VKORC1) in patients with warfarin resistance. From a study group of 820 patients, we identified 4 individuals who required more than 25 mg of warfarin daily for therapeutic anticoagulation.Three of these had serum warfarin concentrations within the therapeutic range of 0.7–2.3 mg/l and showed wild-type VKORC1 sequence. The fourth warfarin resistant individual had consistently high ( ≥ 5.7 mg/l) serum warfarin concentrations, yet had no clinically discernible cause for warfarin resistance. VKORC1 showed a heterozygous 196G→ A transition that predicted aVal66Met substitution in the VKORC1 polypeptide. This transition was also identified in 2 asymptomatic family members who had never received warfarin.These individuals had normal vitamin-K dependent coagulation factor activities and undetectable serum PIVKAII and vitamin K 1 2,3 epoxide suggesting that their basal vitamin K epoxide reductase activity was not adversely affected by the VKORC1 Val66Met substitution.The association between a nucleotide transition in VKORC1 and pharmacodynamic warfarin resistance supports the hypothesis that VKORC1 is the site of action of warfarin and indicates that VKORC1 sequence is an important determinant of the warfarin dose response.

Key Pathways and Regulators of Vitamin K Function and Intermediary Metabolism

2018 ◽  
Vol 38 (1) ◽  
pp. 127-151 ◽  
Author(s):  
Martin J. Shearer ◽  
Toshio Okano
Keyword(s):  
Intestinal Absorption ◽  
Vitamin K ◽  
Intermediary Metabolism ◽  
Biological Functions ◽  
Vitamin K Epoxide Reductase ◽  
Pathophysiological Mechanisms ◽  
Vitamin K Cycle ◽  
Rare Mutations ◽  
Epoxide Reductase ◽  
Key Pathways

Vitamin K (VK) is an essential cofactor for the post-translational conversion of peptide-bound glutamate to γ-carboxyglutamate. The resultant vitamin K–dependent proteins are known or postulated to possess a variety of biological functions, chiefly in the maintenance of hemostasis. The vitamin K cycle is a cellular pathway that drives γ-carboxylation and recycling of VK via γ-carboxyglutamyl carboxylase (GGCX) and vitamin K epoxide reductase (VKOR), respectively. In this review, we show how novel molecular biological approaches are providing new insights into the pathophysiological mechanisms caused by rare mutations of both GGCX and VKOR. We also discuss how other protein regulators influence the intermediary metabolism of VK, first through intestinal absorption and second through a pathway that converts some dietary phylloquinone to menadione, which is prenylated to menaquinone-4 (MK-4) in target tissues by UBIAD1. The contribution of MK-4 synthesis to VK functions is yet to be revealed.

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