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61.
Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors 总被引:94,自引:41,他引:53
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We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport. 相似文献
62.
Biexponential Kinetics of (R)-α-[3 H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor 总被引:3,自引:0,他引:3
Robert E. West Jr. Adam Zweig Russell T. Granzow Marvin I. Siegel Robert W. Egan 《Journal of neurochemistry》1990,55(5):1612-1616
The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated. 相似文献
63.
Adam Bertl 《The Journal of membrane biology》1989,109(1):9-19
Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K+-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K+ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na+ and Cl–. However, addition of Na+ decreases the K+ currents. TheI–V relationships of the open channel as measured at various K+ concentrations with or without Na+ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data. 相似文献
64.
Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly 总被引:42,自引:14,他引:28
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J S Bonifacino C K Suzuki J Lippincott-Schwartz A M Weissman R D Klausner 《The Journal of cell biology》1989,109(1):73-83
The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER. 相似文献
65.
The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis. 相似文献
66.
Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies. 相似文献
67.
68.
David M. Hwang Adam Dempsey Kiat-Tsong Tan Choong-Chin Liew 《Journal of molecular evolution》1996,43(5):536-540
HnifU, a gene exhibiting similarity tonifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a
human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA
verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins fromHaemophilus influenzae andSaccharomyces cerevisiae, respectively, and 40–44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing
organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these
NifU-like proteins exhibited higher sequence identity to each other (63–77%) than to the diazatrophic NifU proteins (40–48%).
Further, the NifU-like proteins of non-nitrogenfixing organisms were similar only to the N-terminal region of diazatrophic
NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis
that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent
as humans andH. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins.
Correspondence to: C.-C. Liew 相似文献
69.
70.