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81.
The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads. 相似文献
82.
A class of ω-aminoalkyl glycosides previously found to antagonize insulin's action on glucose oxidation in fat cells and to stimulate glucose oxidation in insulin's absence is now shown to mimic insulin also on the conversion of glucose to free fatty acids and to glycerol and glycerides. These glycosides also act like insulin by inhibiting hormone- and cholera toxin-stimulated lipolysis. Various lines of evidence demonstrate that most, if not all, of the insulin-like activity of these glycosides results from H2O2 formed from an amine oxidase-catalyzed oxidation of the aminoalkyl moiety of these compounds. A contaminant in the bovine plasma albumin (BPA) preparations used in the bioassays was found to represent a major source of the amine oxidase activity. Membrane (ghost) preparations were also found to possess amine oxidase activity capable of forming H2O2 from the glycosides in amounts sufficient to express insulin-like activity. Preliminary experiments with intact adipocytes suggest that this activity is located on the cell surface. The BPA-associated activity corresponds to the known Cu2+-containing “plasma-type” amine oxidase (EC 1.4.3.6) on the basis of its substrate specificity and susceptibility to selective inhibitors. The plasma membrane activity appears to correspond to neither the plasma-type nor to the flavin-containing mitochondrial-type (EC 1.4.3.4) and remains to be identified. The observed potent antilipolytic effects of both H2O2 and the aminoalkyl glycosides points out that any mechanism used to explain the insulin-like action of H2O2 must account for this ability to inhibit lipolysis as well as to stimulate glucose utilization. That catalase inhibits the insulin-like action of the glycosides and H2O2, but not that of insulin indicates that insulin's action is not mediated by cell surface-produced H2O2. Also, since the insulin antagonistic activity of these glycosides was not inhibited by catalase, H2O2 formation is not responsible for this antagonism. The latter finding, added to present and previous evidence on the carbohydrate structural requirements involved in H2O2 production and in the insulin-like biological and binding properties of the aminoalkyl glycosides, is consistent with a role(s) for their carbohydrate moieties in both the insulin antagonistic and agonistic activities of these compounds. 相似文献
83.
Schwaiger FW; Weyers E; Buitkamp J; Ede AJ; Crawford A; Epplen JT 《Molecular biology and evolution》1994,11(2):239-249
Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for
polymorphisms in both the antigen-binding regions and the adjacent intronic
mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were
identified. Short nucleotide motifs are extensively shared between certain
exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the
number of different amino acids at usually polymorphic sites, and the
number of silent substitutions were reduced in the intraspecies analyses of
sheep DRB sequences, compared with those of cattle and goats. It was
paradoxical that the abundance of different sheep alleles was similar to
that of cattle and goats. This paradox may be explained by postulating a
relatively small number of "ancient" alleles, with the present-day Ovar-DRB
alleles being generated by reciprocal exchange of nucleotide motifs. At the
antigen-binding sites, new combinations of amino acids were maintained in
Ovar-DRB alleles by strong positive selection. In sheep--and less
pronounced in goats and cattle--the DRB alleles can be divided into two
groups. In one group, silent substitutions are increased when compared with
the other. This suggests separate evolutionary pathways for certain groups
of DRB alleles within a species. The simple repetitive sequences are also
discussed with respect to the evolution of DRB alleles.
相似文献
84.
85.
Katzen F Fletcher JE Yang JP Kang D Peterson TC Cappuccio JA Blanchette CD Sulchek T Chromy BA Hoeprich PD Coleman MA Kudlicki W 《Journal of proteome research》2008,7(8):3535-3542
We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets. 相似文献
86.
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89.
We have developed an efficient method for the simultaneous introduction of up to three mutations in a plasmid DNA via homologous recombination. The strategy is compatible with a variety of mutations, including degenerate codons in plasmids of different sizes. In contrast to other methodologies, this approach employs the same set of reagents for both single- and multi-site mutagenesis assays, minimizes the required protocol steps, and exhibits remarkably high mutagenesis efficiencies. 相似文献
90.
David JT. Campbell Paul E. Ronksley Braden J. Manns Marcello Tonelli Claudia Sanmartin Robert G. Weaver Deirdre Hennessy Kathryn King-Shier Tavis Campbell Brenda R. Hemmelgarn for the Interdisciplinary Chronic Disease Collaboration 《PloS one》2014,9(4)