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91.
Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane. 相似文献
92.
Richard M. Napier Michael A. Venis Michael A. Bolton Louise I. Richardson Goeffrey W. Butcher 《Planta》1988,176(4):519-526
Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP
auxin-binding protein
- DEAE
diethylaminoethyl
- Ig
immunoglobulin
- kDa
kilodalton
- NAA
naphthalene-1-acetic acid
- Mr
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
93.
Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells 总被引:3,自引:0,他引:3
Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA. 相似文献
94.
Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed. 相似文献
95.
我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50%,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38%,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。 相似文献
96.
本文报告膜蛋白溶脱剂溶脱大鼠脑M胆碱受体的结果,其中0.5%CHAPS,0.35%洋地黄皂苷和10%甘油的混合液效果较好,可溶脱30%的受体,并得到22%有活性的受体。溶脱的受体有较好的稳定性,与膜结合受体有同样的配体结合特异性,可饱和性及可逆性。平衡结合及动力学研究表明溶脱受体和膜结合受体对[~3H]QNB有类似的亲和性。 相似文献
97.
转铁蛋白结构与功能的研究——骆驼血清转铁蛋自的分离纯化及其含单一铁结合部位的结构域的制备和鉴定 总被引:1,自引:0,他引:1
分离纯化获得的骆驼血清转铁蛋白由分子量为73,000和63,000两个组分组成。两者至少N-端五肽顺序相同(Met-Pro-Asp-Lys-Thr)。骆驼血清转铁蛋白在生理pH下不能与人胎盘转铁蛋白受体结合。用胰蛋白酶酶解骆驼转铁蛋白可以同时得到两个合单一铁结合部位的结构域,分别来自转铁蛋白分子的N-端称N-端结构域(分子量34,700和40,700)和C-端称C-端结构域(分子量35,100)。在上述结果的基础上指出并讨论了反刍动物转铁蛋白在结构和功能上存在更多的共同性,而与其它哺乳动物的转铁蛋白有着明显的区别。 相似文献
98.
本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。 相似文献
99.
Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum 总被引:5,自引:0,他引:5
Richard G. Ham Judy A. St. Clair Cecelia Webster Helen M. Blau 《In vitro cellular & developmental biology. Plant》1988,24(8):833-844
Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized
nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin,
dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS)
as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final
medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo
or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both
SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer
growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little
differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of
increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's
medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were
always those of muscle cells.
This research was supported by a grant from the Muscular, Dystrophy Association.
Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements
for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well
as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy. 相似文献
100.
Charles R. Ill Tammy Brehm Bjorn K. Lydersen Rachel Hernandez Karen G. Burnett 《In vitro cellular & developmental biology. Plant》1988,24(5):413-419
Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific
factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have
been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate
that HxH hybridomas do not respond to bovine transferrin a+ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin.
HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin.
An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH
hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in
HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM
hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth
response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that
the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this
form. 相似文献