Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.     

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH₂). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH₂, and recent studies have called into question whether VKORC1 reduces K to KH₂. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH₂ production. Direct detection of the vitamin K reaction products is confounded by KH₂ oxidation, and we therefore developed a new assay that stabilized KH₂ and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH₂ reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.     

Peculiarities of liver regeneration in Chinese hamster <Emphasis Type="Italic">Cricetulus griseus</Emphasis>

With the aid of cytofluorimetry and interference microscopy, the ploidy level and the hepatocyte ploidy class distribution were studied and the dry mass of hepatocytes was measured in hepatocytes in liver of Chinese hamsters Cricetulus griseus and of Balb/c mice before and one month after partial hepatectomy. The mean ploidy level in hepatocytes of the Chinese hamster normal liver amounted to 2.35 ± 0.03 c. The modal class was mononuclear hepatocytes with diploid nuclei (82.4 ± 1.3%). The mean dry mass of hepatocytes amounted to 605.2 ± 4.8 pg. In the process of liver regeneration in the Chinese hamsters, the ratio of ploidy classes and the hepatocyte dry mass did not change. After a similar liver resection in the mice, a significant polyploidization of liver parenchyma occurred. The mean ploidy level in hepatocytes rose by 32%. Instead of 4cx2-hepatocytes, the modal class became mononuclear octaploid cells the relative portion of which increased, on average, by five times. The portion of binuclear hepatocytes with octaploid nuclei in mouse liver rose by more than five times. Thus, in the Chinese hamsters Cricetulus griseus, unlike mice, regeneration of liver occurred exclusively at the expense of proliferation of hepatocytes.     

Regeneration of the liver of Chinese hamster Cricetulus griseus

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.     

Internal exposure to neutron-activated <Superscript>56</Superscript>Mn dioxide powder in Wistar rats—Part 2: pathological effects

To fully understand the radiation effects of the atomic bombing of Hiroshima and Nagasaki among the survivors, radiation from neutron-induced radioisotopes in soil and other materials should be considered in addition to the initial radiation directly received from the bombs. This might be important for evaluating the radiation risks to the people who moved to these cities soon after the detonations and probably inhaled activated radioactive “dust.” Manganese-56 is known to be one of the dominant radioisotopes produced in soil by neutrons. Due to its short physical half-life, ⁵⁶Mn emits residual radiation during the first hours after explosion. Hence, the biological effects of internal exposure of Wistar rats to ⁵⁶Mn were investigated in the present study. MnO₂ powder was activated by a neutron beam to produce radioactive ⁵⁶Mn. Rats were divided into four groups: those exposed to ⁵⁶Mn, to non-radioactive Mn, to ⁶⁰Co γ rays (2 Gy, whole body), and those not exposed to any additional radiation (control). On days 3, 14, and 60 after exposure, the animals were killed and major organs were dissected and subjected to histopathological analysis. As described in more detail by an accompanying publication, the highest internal radiation dose was observed in the digestive system of the rats, followed by the lungs. It was found that the number of mitotic cells increased in the small intestine on day 3 after ⁵⁶Mn and ⁶⁰Co exposure, and this change persisted only in ⁵⁶Mn-exposed animals. Lung tissue was severely damaged only by exposure to ⁵⁶Mn, despite a rather low radiation dose (less than 0.1 Gy). These data suggest that internal exposure to ⁵⁶Mn has a significant biological impact on the lungs and small intestine.