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2.
Affinity purification of the HIV-1 protease 总被引:5,自引:0,他引:5
J C Heimbach V M Garsky S R Michelson R A Dixon I S Sigal P L Darke 《Biochemical and biophysical research communications》1989,164(3):955-960
An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column. 相似文献
3.
Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21. 相似文献
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G E Rukavishnikova M I Potapov E R Sigal 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1985,(1):69-73
The use of the indirect ELISA techniques did not ensure the sharp differentiation of the antigens of the blood groups A and B on the polystyrene sorbent by means of heteroimmune sera, though such differentiation could be achieved by means of monoclonal antibodies. The test system known as "the lectin-antibody sandwich" was found to have the optimum sensitivity and specificity permitting the detection of soluble ABH antigens. This variant of ELISA permitted the detection of blood group A antigen both in native biological materials and in traces of blood and saliva, thus making it possible to carry out its quantitative determination. 相似文献
6.
Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells 总被引:4,自引:0,他引:4
L D Stein C J Ledgley N H Sigal 《Journal of immunology (Baltimore, Md. : 1950)》1983,130(4):1640-1645
The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro. 相似文献
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Ras interaction with the GTPase-activating protein (GAP) 总被引:18,自引:0,他引:18
M D Schaber V M Garsky D Boylan W S Hill E M Scolnick M S Marshall I S Sigal J B Gibbs 《Proteins》1989,6(3):306-315
Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32-40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser-35]Ras binds to GAP as effectively as wild-type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser-35]Ras can also efficiently stimulate the S. cerevisiae target of Ras, adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17-44 and 17-32 competed with the binding of Ras to E. coli-expressed GAP with IC50 values of 2.4 and 0.9 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17-32 may be important for Ras binding to GAP. 相似文献
9.
A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum 总被引:8,自引:0,他引:8 下载免费PDF全文
In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn2+.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn2+.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site. 相似文献
10.
Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells. 相似文献