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131.
Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit 总被引:5,自引:0,他引:5
J R Emanuel J Schulz X M Zhou R B Kent D Housman L Cantley R Levenson 《The Journal of biological chemistry》1988,263(16):7726-7733
We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells. 相似文献
132.
Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution 总被引:6,自引:0,他引:6
P G Schoofs H M Geysen D C Jackson L E Brown X L Tang D O White 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(2):611-616
Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues. 相似文献
133.
本文对受到临界全致死剂量——8.5Gy~(60)Co照射后的小鼠造血干细胞(HSC)的自我更新力进行了测试,并对照射后4个月,造血功能业已恢复的小鼠HSC的造血重建功能进行了研究。结果表明:应用骨髓连续移植实验所测得的照后3个月中骨髓CFU_s的自我更新潜能明显衰退了。用照射后造血恢复小鼠的CFU_s给全致死剂量照射的受体进行移植治疗,发现其移植效力比正常的显著减弱。在受体存活30天时,CFU_s的再生速率只有正常的1/17。通过对性染色体追踪观察的资料分析,此种小鼠的??造血细胞在受体中难以形成长期稳定的嵌合体。以上事实反映出,照射后小鼠的HSC的造血重建功能大大削弱了,揭示出残存干细胞质量上的缺陷。对于这种潜在的残留损伤的机制,从“干细胞增殖力耗竭学说”和“基因自我修整假说”的角度进行了讨论。 相似文献
134.
X Bertagna F Camus F Lenne F Girard J P Luton 《Molecular endocrinology (Baltimore, Md.)》1988,2(11):1108-1114
The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules. 相似文献
135.
A cell type-specific enhancer drives expression of the chick muscle acetylcholine receptor alpha-subunit gene 总被引:14,自引:0,他引:14
The regulation of acetylcholine receptor alpha-subunit gene expression was analyzed by transient expression assays. Using rabbit beta-globin cDNA as a reporter gene, we have confirmed that the 5'-flanking sequence of the chicken acetylcholine receptor alpha-subunit gene directs specific expression in differentiated C2C12 cells, a mouse muscle cell line, but not in undifferentiated C2C12 cells and mouse 3T3 fibroblasts. Testing chimeric plasmids containing Bal31 deletion mutants of the alpha-subunit gene upstream sequence, we found the -116 to -81 region of the alpha-subunit to be responsible for tissue- and stage-specific expression. This 36 bp fragment stimulates the activity of both alpha-subunit and SV40 promoters in a distance- and orientation-independent manner, thus fulfilling the criteria of an enhancer. 相似文献
136.
The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum. Both proteins contain one selenocysteine and two cysteine residues. 相似文献
137.
Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa
Summary
Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores. 相似文献
138.
作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。 相似文献
139.
A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold. 总被引:23,自引:0,他引:23 下载免费PDF全文
Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity. 相似文献
140.