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31.
Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases. 下载免费PDF全文
T. Kitlar F. Dring D. F. Diedrich R. Frank H. Wallmeier R. K. Kinne J. Deutscher 《Protein science : a publication of the Protein Society》1994,3(4):696-700
Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity. 相似文献
32.
Sandra L. Gage Stanley R. Keim Jay R. Simon Walter C. Low 《Neurochemical research》1994,19(11):1379-1386
The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC. 相似文献
33.
S. H. Ogo C. F. Bernardes M. L. Glass M. A. Torsoni A. E. Vercesi 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(7):614-619
Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca2+ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca2+ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O2 affinity, which suggests that ATP production by mitochondria may participate in modulation of O2 transport by hemoglobin.Abbreviations
membrane potential
- BAE
Bothrops alternatus erythrocytes
- DNP
2,4-dinitrophenol
- DPG
2,3-diphosphoglycerate
- EGTA
ethyleneglycol tetra-acetic acid
- FCCP
carbonylcyanide p-trifloromethoxyphenylhydrazone
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine
- TPP+
tetraphenylphosphonium
- TRIS
tris-(hydroxymethyl)aminomethane 相似文献
34.
A chemically reactive analog of the phytotropin N-1-naphthylphthalamic acid (NPA) was synthesized and evaluated as a site-directed irreversible ligand for the NPA receptor. The NPA analog (5-isothiocyanato-N-1-naphthylphthalamic acid; NCS-NPA) was synthesized in two steps. Pretreatment of etiolated Helianthus hypocotyl segments with NCS-NPA at concentrations in excess of 1 M resulted in a dose-dependent inhibition of basipetal [14C]IAA transport. Net uptake of IAA by hypocotyl segments was stimulated by NCS-NPA at concentrations of 1 M or greater. NCS-NPA inhibited the saturable binding of [3H]NPA in Helianthus microsomes in a dose-dependent fashion with 50% inhibition occurring at NCS-NPA concentrations of 3 to 10 nM. The binding affinity of [3H]NPA in microsomes pretreated with NCS-NPA followed by extensive washing was substantially reduced. These results demonstrate that NCS-NPA is a site-directed irreversible ligand for the NPA receptor and suggest that it may be of use in the purification and characterization of this biologically important receptor.Abbreviations ANPA
5-amino-naphthylphthalamic acid
- IAA
indole-3-acetic acid
- NCS-NPA
5-isothiocyanato-N-1-naphthylphthalamic acid
- NPA
N-1-naphthylphthalamic acid
- TLC
thin-layer chromatography 相似文献
35.
Albert J. Parker 《Plant Ecology》1994,115(2):145-155
Latitudinal gradients of tree species composition along the Sierran/Cascade axis in northern California were explored by comparing forests of Lassen Volcanic and Yosemite National Parks, USA. A calibration procedure based on canonical correspondence analysis predicted a mean rate of elevational displacement of 172.1 m/° latitude for Lassen sites in Yosemite. This is a steep latitudinal gradient compared with other temperate uplands (which average around 100 m/0 latitude), but it corresponds with the magnitude of the July mean temperature gradient (143 m/0 latitude) and the annual precipitation gradient (230 m/0 latitude). Elevational displacement of basal-area weighted species means showed considerable variation. The range for montane species was 20–153 m/0 latitude; for subalpine species the range was 142–305 m/0 latitude. This disparity is related to differential temperature lapse rates between regions and is reinforced by contrasting biogeographic affinities of montane vs. subalpine species. Whereas it is uniformly hot and dry during the growing season at lower elevations in both regions, growing seasons in the subalpine zone are significantly warmer and drier (at comparable elevations) in Yosemite, the more southerly locale. Furthermore, montane species are principally of Sierran affinity, whereas subalpine are primarily of Pacific Northwestern affinity. 相似文献
36.
利用标记N-糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯(PMA)对人肝癌细胞SMMC-7721表面糖蛋白上N-糖链结构的影响,发现100nmol/L的PMA处理5天后,可使细胞表面N-糖链中高甘露糖型和杂合型以及四天线、C2C2,6三天线复杂型的比例增高,而二天线复杂型降低。此结果与我们曾报道的视黄酸(RA)和双丁配环磷酸腺苷(db-cAMP)对该细胞表面N-糖链的影响相反。因RA和db-cAMP是SMMC-7721细胞的分化诱导剂,可抑制细胞生长;而PMA是该细胞的增殖促进剂,故细胞表面N-糖链的变化与细胞的分化和增殖密切相关。 相似文献
37.
A rapid two-step procedure has been developed for the purification of Despro(2)-Val15-Leu17-aprotinin from the culture supernatant of a recombinant yeast by affinity and ion-exchange chromatography. DesPro(2)-Val15-Leu17-aprotinin was purified to homogeneity, as demonstrated by dodecylsulfate gel electrophoresis and analysis of the N-terminal amino acid sequence. (c) 1993 John Wiley & Sons, Inc. 相似文献
38.
平菇萃取液经酸性沉淀、热变性、Phenyl-Sepharose CL-4B疏水亲和层析和DEAE-Cellulose 52离子交換层析分冉出了电泳纯的真菌钙调素。在比较钙调素对磷酸二酯酶活化的能力和ELISA实验的免疫亲和力对发现,平菇钙调素与猪脑钙调素的生物活性有较大差异,提示在钙调素定量测定中有必要考虑到标准钙调素与样品钙调素之间的同源性差异。 相似文献
39.
本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸(RA)作用1—5天过程中人肝癌细胞株SMMC-7721细胞表面N糖链结构的变化。结果表明,RA促进3~H-甘露糖(Man)参入细胞表面N糖链,使高甘露糖型N糖链的百分比下降,复杂型百分比上升,并促进二天线N糖链的生物合成,使多天线特别是四天线和C_2,C_(21)b三天线N糖链的合成减少。结果提示,N糖链结构的这些变化可能是RA诱导SMMC-7721细胞向正常方向分化的结果。 相似文献
40.
E. Mark Williams Mogens L. Glass Norbert Heisler 《Environmental Biology of Fishes》1993,37(4):407-413
Synopsis The oxygen transport characteristics and the acid-base status of carp blood was studied in vitro by equilibration of blood samples with and without addition of 5mmol l–1 of nitrite for 30 min at various Po2 values in combination with Pco2 of 1.5 and 5.7mmHg (0.2 and 0.76kPa). After equilibration pH, Po2, Pco2, and Co2 as well as methaemoglobin and HCO3
– concentration were determined and oxygen dissociation curves established. At Pco2 of 1.5mmHg (0.2kPa) oxygen affinity, expressed by a normal P50 of 3.3mmHg (0.44kPa) was unaffected by nitrite exposure, whereas at Pco2 5.7 (0.76kPa), nitrite exposure shifted P50 from 7.59mmHg (1.01kPa) to 21.9mmHg (2.92kPa). Methaemoglobin formation was greater at the higher Pco2 and increased with falling Po2. Erythrocyte shrinkage and rising plasma [HC03
–] during nitrite exposure indicated that the erythrocyte osmoregulation was significantly affected. The present results indicate significantly reduced oxygen affinity upon exposure of carp blood to nitrite. This result contrasts with findings in mammalian blood, where oxygen affinity is greatly enhanced. 相似文献