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171.
172.
Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5 总被引:1,自引:0,他引:1
Altuve A Silchenko S Lee KH Kuczera K Terzyan S Zhang X Benson DR Rivera M 《Biochemistry》2001,40(32):9469-9483
Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5. 相似文献
173.
Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects 总被引:2,自引:0,他引:2 下载免费PDF全文
The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel. 相似文献
174.
175.
Nitric oxide production, nitric oxide synthase (NOS) and mitochondrial nitrite-reducing activities in roots, leaves and stems
of different developmental stages were investigated, using potted 3-year-old apple (Malus domestica Borkh.) trees. The arginine-dependent NOS activity is sensitive to NOS inhibitor L-NAME and aminoguanidine (AG), with L-NAME
being more effective than AG. Endogenous NO production, NOS and mitochondrial nitrite-reducing activities are predominately
presented in young leaves and especially in young white roots and young stems. Root and stem mitochondria can reduce nitrite
to nitric oxide at the expense of NADH, however, this mitochondrial nitrite-reducing activity is absent in leaves. 相似文献
176.
177.
178.
Leaf mass per unit area (LMA), carbon and nitrogen contents, leaf construction cost, and photosynthetic capacity (P
max) of Adiantum reniforme var. sinensis, an endangered fern endemic to the Three Gorges region in southwest China, were compared in five populations differing in
habitat such as soil moisture and irradiance. The low soil moisture and high irradiance habitat population exhibited significantly
higher LMA, area-based leaf construction (CCA), and carbon content (CA), but lower leaf nitrogen content per unit dry mass (NM) than the other habitat populations. The high soil moisture and low irradiance habitat populations had the lowest CCA, but their cost/benefic ratios of CCA/P
max were similar to the medium soil moisture and irradiance habitat population due to their lower leaf P
max. Hence A. reniforme var. sinensis prefers partially shaded, moist but well-drained, slope habitats. Due to human activities, however, its main habitats now
are cliffs or steeply sloped bare rocks with poor and thin soil. The relatively high energy requirements and low photosynthetic
capacity in these habitats could limit the capability of the species in extending population or interspecific competition
and hence increase its endangerment. 相似文献
179.
Sachs PC Francis MP Zhao M Brumelle J Rao RR Elmore LW Holt SE 《Cell and tissue research》2012,349(2):505-515
The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location. 相似文献
180.
Redox-active iron mediates amyloid-beta toxicity 总被引:12,自引:0,他引:12
Rottkamp CA Raina AK Zhu X Gaier E Bush AI Atwood CS Chevion M Perry G Smith MA 《Free radical biology & medicine》2001,30(4):447-450
While amyloid-beta toxicity is mediated by oxidative stress and can be attenuated by antioxidants, the actual biochemical mechanism underlying neurotoxicity remains to be established. However, since aggregated amyloid-beta can interact with transition metals, such as iron, both in vitro and in vivo, we suspected that bound iron might be the mediator of toxicity such that holo- and apo-amyloid would have differential effects on cellular viability. Here we demonstrate that when amyloid-beta is pretreated with the iron chelator deferoxamine, neuronal toxicity is significantly attenuated while conversely, incubation of holo-amyloid-beta with excess free iron restores toxicity to original levels. These data, taken together with the known sequelae of amyloid-beta, suggest that the toxicity of amyloid-beta is mediated, at least in part, via redox-active iron that precipitates lipid peroxidation and cellular oxidative stress. 相似文献