Lack of Association Between Type 2 Diabetes and the 3673G / A and 9041G / A Gene Variants of Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1)

2016 ◽  
Vol 86 (3-4) ◽  
pp. 133-139
Author(s):  
Cansu Ozbayer ◽  
Hulyam Kurt ◽  
Medine Nur Kebapci ◽  
Didem Turgut Cosan ◽  
Ertugrul Colak ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Vitamin K ◽  
Control Group ◽  
Gene Variants ◽  
Vitamin K Epoxide Reductase ◽  
Vitamin K Cycle ◽  
Genotype Frequencies ◽  
Epoxide Reductase ◽  
Vkorc1 Gene

The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene is expressed in many tissue types, and encodes the VKORC1 protein, which is a key enzyme in the vitamin K cycle. Although researchers have focused on the effects of vitamin K on glucose metabolism, and on its role in the development of type 2 diabetes (T2DM), no consensus has yet been reached. Therefore, here we aimed to investigate the association between VKORC1 variants and the risk of T2DM. The 3673G / A (rs9923231) and 9041G / A (rs7294) VKORC1 variants were investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 control individuals and 100 patients with T2DM. The genomic regions were amplified by PCR; amplicons were digested using the AciI and NciI enzymes and visualized by agarose gel electrophoresis. The genotype frequencies of the 3673G / A variants were GG (22%), GA (56%), and AA (22%) in the control group and GG (19%), GA (52%), and AA (29%) in patients with T2DM (p > 0.05). The genotype frequencies of the 9041G / A variants were GG (37%), GA (53%), and AA (10%) in the control group and GG (46%), GA (45%), and AA (9%) in patients with T2DM (p > 0.05). In conclusion, we found no significant correlation between the control group and patients with T2DM, with regard to the different genetic models of the 3673G / A and 9041G / A variants. These data suggest that these VKORC1 gene variants may not be linked to T2DM.

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.

Vitamin K epoxide reductase (VKORC1) gene mutations in osteoporosis: A pilot study

2010 ◽  
Vol 156 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Gerold Holzer ◽  
Anna Verena Grasse ◽  
Sonja Zehetmayer ◽  
Peter Bencur ◽  
Christian Bieglmayer ◽  
...  
Keyword(s):  
Pilot Study ◽  
Vitamin K ◽  
Gene Mutations ◽  
Vitamin K Epoxide Reductase ◽  
Epoxide Reductase ◽  
Vkorc1 Gene

Haplogroup analysis of vitamin-K epoxide reductase (VKORC1) gene: novel element in the optimization of anticoagulant therapy

2008 ◽  
Vol 149 (39) ◽  
pp. 1839-1844
Author(s):  
Csilla Sipeky ◽  
Béla Melegh
Keyword(s):  
Anticoagulant Therapy ◽  
Vitamin K ◽  
Vitamin K Epoxide Reductase ◽  
Pcr Rflp ◽  
Epoxide Reductase ◽  
Vkorc1 Gene

A warfarin és az acenokumarolok a leggyakrabban alkalmazott antikoagulánsok, amelyek szűk terápiás tartománnyal rendelkeznek, a hatásos dózis pedig populáción belül és egyénenként is nagy változatosságot mutat. A kumarinok a K-vitamin-epoxidreduktáz enzim (VKOR) gátlásán keresztül akadályozzák meg a koagulációt. Az enzimet kódoló VKORC1 gén mutációi jelentősen befolyásolják a kumarinok iránti érzékenységet. A VKORC1 gén genetikai variabilitását a *2, *3 és a *4 haplotípusok fedik le a kaukázusi populációban. Antikoaguláns kezelésben részesülő betegek bemutatásán keresztül összefoglaló tanulmányban ismertetjük a VKORC1 gén haplotípusának variabilitását. Munkánkban 28, klinikailag nem szokványos antikoaguláns választ produkáló beteget karakterizáltunk a VKORC1 G-1639A, G9041A és C6009T polimorfizmusokra. Molekuláris módszerként PCR-RFLP technikát és direkt szekvenálást alkalmaztunk. Betegpopulációnkban sikerült kimutatni VKORC1 *1*2, *2*2, *2*3, *1*4, *2*4 és *3*4 haplotípusokat. Vizsgált betegeink körében előfordult a VKORC1 gén haplotípusa alapján közepes dózisigényű (4,9±0,2 mg/nap) A/B haplocsoportú (a vizsgált betegek 61%-a) és magas dózisigényű (6,2±0,3 mg/nap) B haplocsoportú (25%) beteg is. Az antikoaguláns terápia vérzéses szövődményeinek megelőzésében fontos az alacsony warfarindózisú (2,7±0,2 mg/nap) A haplocsoportba tartozó betegek (esetünkben 14%) diagnosztizálása. Eredményeink mutatják, hogy a haplocsoport-vizsgálat segíti a megfelelő szintű véralvadásgátláshoz szükséges gyógyszerdózis meghatározását és a végzetes vérzési epizódok elkerülését.

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.

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