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131.
132.
Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy. 相似文献
133.
The reactivity and relative rarity of most cofactors pose challenges for their delivery to target enzymes. Using kinetic analyses, we demonstrate that adenosyltransferase, which catalyzes the final step in the assimilation of coenzyme B12, directly transfers the cofactor to methylmalonyl coenzyme A mutase. The strategy of using the final enzyme in an assimilation pathway for tailoring a cofactor and delivering it to a dependent enzyme may be general for cofactor trafficking. 相似文献
134.
Human cystathionine β-synthase (CBS) catalyzes the first irreversible
step in the transsulfuration pathway and commits homocysteine to the synthesis
of cysteine. Mutations in CBS are the most common cause of severe hereditary
hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that
interact with CBS had previously identified several components of the
sumoylation pathway and resulted in the demonstration that CBS is a substrate
for sumoylation. In this study, we demonstrate that sumoylation of CBS is
enhanced in the presence of human polycomb group protein 2 (hPc2), an
interacting partner that was identified in the initial yeast two-hybrid screen.
When the substrates for CBS, homocysteine and serine for cystathionine
generation and homocysteine and cysteine for H2S generation, are
added to the sumoylation mixture, they inhibit the sumoylation reaction, but
only in the absence of hPc2. Similarly, the product of the CBS reaction,
cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn
decreases CBS activity by ∼28% in the absence of hPc2 and by
70% in its presence. Based on these results, we conclude that hPc2
serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We
also demonstrate that γ-cystathionase, the second enzyme in the
transsulfuration pathway is a substrate for sumoylation under in vitro
conditions. We speculate that the role of this modification may be for nuclear
localization of the cysteine-generating pathway under conditions where nuclear
glutathione demand is high. 相似文献
135.
136.
BACKGROUND: Rosai-Dorfman disease (RDD), or sinus histiocytosis with massive lymphadenopathy, is a benign, self-limiting histiocytic proliferative disorder commonly involving the lymph nodes. Extranodal disease occurs in about 40% of cases, and the sites involved are skin, nasal cavity, paranasal sinuses, eyelids, orbit, bone and central nervous system. CASE: A case of RDD presented as subcutaneous nodules and was diagnosed on a fine needle aspirate. The aspirate revealed numerous histiocytes with phagocytosis of lymphocytes, plasma cells and neutrophils. Surgical biopsy and immunocytochemical stain for S-100 protein confirmed the diagnosis. Later the patient developed lymphadenopathy and involvement of the nasal septum. CONCLUSION: Extranodal RDD can be diagnosed by FNAC in conjunction with immunocytochemistry. 相似文献
137.
Pal-Ghosh R Yu J Prado GN Taylor L Mierke DF Polgar P 《Archives of biochemistry and biophysics》2003,415(1):54-62
The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake. 相似文献
138.
Mansoorabadi SO Padmakumar R Fazliddinova N Vlasie M Banerjee R Reed GH 《Biochemistry》2005,44(9):3153-3158
The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme. 相似文献
139.
Narayanan S Ruma D Gitika B Sharma SK Pauline T Ram MS Ilavazhagan G Sawhney RC Kumar D Banerjee PK 《Molecular and cellular biochemistry》2005,278(1-2):9-14
The present study reports the cytoprotective and antioxidant properties of alcoholic leaf extract of seabuckthorn (SBT) against
hypoxia induced oxidative stress in C-6 glioma cells. Exposure of cells to hypoxia for 12 h resulted in a significant increase
in cytotoxicity and decrease in mitochondrial transmembrane potential compared to the controls. Further an appreciable increase
in nitric oxide and reactive oxygen species (ROS) production was noted which in turn was responsible for fall in intracellular
antioxidant levels and GSH/GSSG ratio. There was a significant increase in DNA damage during hypoxia as revealed by comet
assay. Pretreatment of cells with alcoholic leaf extract of SBT at 200 μg/ml significantly inhibited cytotoxicity, ROS production
and maintained antioxidant levels similar to that of control cells. Further, the leaf extract restored the mitochondrial integrity
and prevented the DNA damage induced by hypoxia. These results indicate that the leaf extract of SBT has strong antioxidant
and cytoprotective activity against hypoxia induced oxidative injury. (Mol Cell Biochem 278: 9–14, 2005) 相似文献
140.
Methionine synthase reductase (MSR) catalyzes the conversion of the inactive form of human methionine synthase to the active state of the enzyme. This reaction is of paramount physiological importance since methionine synthase is an essential enzyme that plays a key role in the methionine and folate cycles. A common polymorphism in human MSR has been identified (66A --> G) that leads to replacement of isoleucine with methionine at residue 22 and has an allele frequency of 0.5. Another polymorphism is 524C --> T, which leads to the substitution of serine 175 with leucine, but its allele frequency is not known. The I22M polymorphism is a genetic determinant for mild hyperhomocysteinemia, a risk factor for cardiovascular disease. In this study, we have examined the kinetic properties of the M22/S175 and I22/S175 and the I22/L175 and I22/S175 pairs of variants. EPR spectra of the semiquinone forms of variants I22/S175 and M22/S175 are indistinguishable and exhibit an isotropic signal at g = 2.00. In addition, the electronic absorption and reduction stoichiometries with NADPH are identical in these variants. Significantly, the variants activate methionine synthase with the same V(max); however, a 3-4-fold higher ratio of MSR to methionine synthase is required to elicit maximal activity with the M22/S175 and I22/L175 variant versus the I22/S175 enzyme. Differences are also observed between the variants in the efficacies of reduction of the artificial electron acceptors: ferricyanide, 2,6-dichloroindophenol, 3-acetylpyridine adenine dinucleotide phosphate, menadione, and the anticancer drug doxorubicin. These results reveal differences in the interactions between the natural and artificial electron acceptors and MSR variants in vitro, which are predicted to result in less efficient reductive repair of methionine synthase in vivo. 相似文献