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51.
Although the peptide C(alpha)H group has historically not been thought to form hydrogen bonds within proteins, ab initio quantum calculations show it to be a potent proton donor. Its binding energy to a water molecule lies in the range between 1.9 and 2.5 kcal/mol for nonpolar and polar amino acids; the hydrogen bond (H-bond) involving the charged lysine residue is even stronger than a conventional OH..O interaction. The preferred H-bond lengths are quite uniform, about 3.32 A. Formation of each interaction results in a downfield shift of the bridging hydrogen's chemical shift and a blue shift in the C(alpha)H stretching frequency, potential diagnostics of the presence of such an H-bond within a protein. 相似文献
52.
Singh Anjana Pandey K.D. Dubey R.S. 《World journal of microbiology & biotechnology》1999,15(2):277-282
Reverse micelles serve as a novel tool to entrap enzymes and microbial whole cells within aqueous pockets and can be of great use in enhancing the efficiency and sustainability of the biological system. Photosynthetic bacterium Rhodopseudomonas sphaeroides entrapped inside the aqueous pool of reverse micelles prepared from benzene-sodium lauryl sulphate exhibited 25-fold enhancement of H2 photoproduction rate (1.67 ml H2 [mg protein]–1 h–1) compared to cells suspended in normal aqueous medium. Hydrogen photoproduction by the bacterium was catalysed by the nitrogenase enzyme system which was supported at a low light intensity of 12 Em–2 sec–1 photon flux energy at a wavelength of 520 nm. The optimum temperature for the process was 40 °C. 相似文献
53.
Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes. 相似文献
54.
Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat 总被引:1,自引:0,他引:1
Rhee MS Perianayagam A Chen P Youn JH McDonough AA 《American journal of physiology. Cell physiology》2004,287(5):C1229-C1237
Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na+-K+-ATPase (NKA) isoform proteins found in muscle, 2 and 1, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K+ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg·kg1·day1 for 14 days (low DEX; n = 5 pairs) or 0.1 mg·kg1·day1 for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU·kg1·min1 over 2.5 h in conscious rats. Insulin-stimulated cellular K+ and glucose uptake rates were assessed in vivo by measuring the exogenous K+ infusion () and glucose infusion (Ginf) rates needed to maintain constant plasma K+ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. Ginf (in mmol·kg1·h1) was 10.2 ± 0.6 in vehicle-treated rats, 5.8 ± 0.8 in low-DEX-treated rats, and 5.2 ± 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K+ uptake. (in mmol·kg1·h1) was 0.53 ± 0.08 in vehicle-treated rats, 0.49 ± 0.14 in low-DEX-treated rats, and 0.27 ± 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K+ excretion. NKA 2-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 ± 15% (soleus) and by 67 ± 3% (gastrocnemius) in the high-DEX treatment group. The NKA 1-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K+ clearance during chronic DEX treatment. Insulin-stimulated cellular K+ uptake was significantly depressed despite increased muscle sodium pump pool size. skeletal muscle; sodium pump; Na+-K+-ATPase 相似文献
55.
56.
Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO2 and GMbO2, respectively. H2O2-induced iron release was significantly higher from GMbO2 than that from MbO2. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H2O2 was studied. GMbO2 degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO2. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H2O2 with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO2. Compared to MbO2, GMbO2 was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO2. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H2O2. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients. 相似文献
57.
El-Sayed NM Ghedin E Song J MacLeod A Bringaud F Larkin C Wanless D Peterson J Hou L Taylor S Tweedie A Biteau N Khalak HG Lin X Mason T Hannick L Caler E Blandin G Bartholomeu D Simpson AJ Kaul S Zhao H Pai G Van Aken S Utterback T Haas B Koo HL Umayam L Suh B Gerrard C Leech V Qi R Zhou S Schwartz D Feldblyum T Salzberg S Tait A Turner CM Ullu E White O Melville S Adams MD Fraser CM Donelson JE 《Nucleic acids research》2003,31(16):4856-4863
58.
The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6. 相似文献
59.
Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver 总被引:13,自引:0,他引:13
Van Eck M Twisk J Hoekstra M Van Rij BT Van der Lans CA Bos IS Kruijt JK Kuipers F Van Berkel TJ 《The Journal of biological chemistry》2003,278(26):23699-23705
Scavenger receptor class B, type I (SRBI) is a key regulator of high density lipoprotein (HDL) metabolism. It facilitates the efflux of cholesterol from cells in peripheral tissues to HDL and mediates the selective uptake of cholesteryl esters from HDL in the liver. We investigated the effects of SRBI deficiency in the arterial wall and in the liver using SRBI-deficient mice and wild-type littermates fed a Western-type diet. The SRBI-deficient mice showed massive accumulation of cholesterol-rich HDL in the circulation, reflecting impaired delivery to the liver. Strikingly, SRBI deficiency did not alter hepatic cholesterol (ester) content nor did it affect the expression of key regulators of hepatic cholesterol homeostasis, including HMG-CoA reductase, the low density lipoprotein receptor, and cholesterol 7alpha-hydroxylase. However, a approximately 40% reduction in biliary cholesterol content was observed, and the expression of ABCG8 and ABCG5, ATP half-transporters implicated in the transport of sterols from the liver to the bile, was attenuated by 70 and 35%, respectively. In contrast to the situation in the liver, SRBI deficiency did result in lipid deposition in the aorta and atherosclerosis. Vascular mRNA analysis showed increased expression of inflammatory markers as well as of genes involved in cellular cholesterol homeostasis. Our data show that, although hepatic cholesterol homeostasis is maintained upon feeding a Western-type diet, SRBI deficiency is associated with de-regulation of cholesterol homeostasis in the arterial wall that results in an increased susceptibility to atherosclerosis. 相似文献
60.
Hoekstra M Kruijt JK Van Eck M Van Berkel TJ 《The Journal of biological chemistry》2003,278(28):25448-25453
Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI ( approximately 3-fold), peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma (8-20-fold), cholesterol 7alpha-hydroxylase A1 (>100-fold), and ABCG5/G8 ( approximately 5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)beta, PPARdelta, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRalpha, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level. 相似文献