| 2374926 |
The structure of a complex of recombinant hirudin and human alpha-thrombin |
10.1126/science.2374926. |
Science |
The structure of a complex of recombinant hirudin and human alpha-thrombin
Abstract
- The crystallographic structure of a recombinant hirudin-thrombin complex has been solved at 2.3 angstrom (A) resolution. Hirudin consists of an NH2-terminal globular domain and a long (39 A) COOH-terminal extended domain. Residues Ile1 to Tyr3 of hirudin form a parallel beta-strand with Ser214 to Glu217 of thrombin with the nitrogen atom of Ile1 making a hydrogen bond with Ser195 O gamma atom of the catalytic site, but the specificity pocket of thrombin is not involved in the interaction. The COOH-terminal segment makes numerous electrostatic interactions with an anion-binding exosite of thrombin, whereas the last five residues are in a helical loop that forms many hydrophobic contacts. In all, 27 of the 65 residues of hirudin have contacts less than 4.0 A with thrombin (10 ion pairs and 23 hydrogen bonds). Such abundant interactions may account for the high affinity and specificity of hirudin.
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| 2376472 |
Synthesis and immunosuppressive activities of conformationally restricted cyclosporin lactam analogues |
10.1111/j.1399-3011.1990.tb00076.x. |
Int J Pept Protein Res |
Synthesis and immunosuppressive activities of conformationally restricted cyclosporin lactam analogues
Abstract
- Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, is known to adopt predominantly a single conformation in chloroform solution, characterized in part by a type II' beta-turn encompassing Abu-Sar-MeLeu-Val (residues 2-5). In order to evaluate whether this beta-turn is bound by the receptor, we previously had prepared a conformationally restricted beta-turn analogue, (delta-lactam) CsA (2), which was found to retain only weak immunosuppressive activity, a result that could indicate that steric hindrance between receptor and the lactam atoms in 2 diminished activity or that the type II' beta-turn is not a feature in the bioactive conformation of CsA. In an attempt to distinguish between these two possibilities, we have synthesized two new CsA analogues, (gamma-lactam) CsA (3) and (des-N-methyl-lactam) CsA (4), which contain less sterically demanding conformational restrictions. The immunosupressive activity of each analogue (4-13% and 7-17%, respectively, relative to CsA), measured in an assay that determined the inhibition of concanavalin A stimulated thymocytes, is essentially equipotent with that of the delta-lactam. The chemical shifts and temperature dependencies of the protons in analogues 3 and 4 are very similar to the corresponding protons in CsA and in 2, which suggest that the solution conformations of the small lactam analogues are very similar to that of the delta-lactam 2. The synthesis of the lactam components and the corresponding CsA derivatives is described. Reduction in the size of the lactam ring does not lead to enhanced immunosuppressive activity."
|
| 2389255 |
Cyclosporine A inhibition of microcystin toxins |
10.1016/0041-0101(90)90301-m. |
Toxicon |
Cyclosporine A inhibition of microcystin toxins
Abstract
- Cyclosporine A (CyA) given i.v. at a dose of 1.25 mg/mouse blocks a subsequent i.v. lethal dose (1.7-1.8 x LD50) of microcystin-LR for 24 hr, and is about 50% protective at 48 hr. Conversely, the fraction of mice that can be rescued by CyA (0.2 mg/mouse) after a lethal dose of microcystin-LR decreases rapidly with a pharmacodynamic half-time of only about 100 sec. The prophylactic action of CyA was tested against lethal doses of four microcystins. The acute lethality of 1.7-1.8 x LD50 dose of microcystin-LR, -RR, -LY, or -LA given 1 hr after administration of 0.2 mg of CyA is 0%, 0%, 58%, or 100%, respectively. Even a 0.6 mg/mouse dose of CyA is ineffective prophylaxis against a lethal dose of microcystin-LA. The inhibitory potency of CyA on microcystin toxicity can be completely reversed by the single L-amino acid substitution of alanine for arginine in the microcystin.
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| 2392682 |
A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation |
10.1126/science.2392682. |
Science |
A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation
Abstract
- The ligand-binding function of integrin adhesion receptors depends on divalent cations. A mutant alpha IIb beta 3 integrin (platelet gpIIb/IIIa) that lacks ligand recognition shows immunologic evidence of a perturbed interaction with divalent cations. This was found to be caused by a G----T mutation that resulted in an Asp119----Tyr119 substitution in the beta 3 subunit. This residue is proximal to bound ligand and is in a conserved region among integrins that are enriched in oxygenated residues. The spacing of these residues aligns with the calcium-binding residues in EF hand proteins, suggesting interaction with receptor-bound divalent cation as a mechanism of ligand binding common to all integrins.
|
| 2397216 |
Epidermal growth factor labeled beta-amanitin-poly-L-ornithine: preparation and evidence for specific cytotoxicity |
10.1021/bi00481a012. |
Biochemistry |
Epidermal growth factor labeled beta-amanitin-poly-L-ornithine: preparation and evidence for specific cytotoxicity
Abstract
- Poly-L-ornithine with an average molecular weight of 32K was reacted with beta-amanitin hydroxysuccinimide ester to form an amide-linked toxin conjugate. Loading of the polymeric chain with amanitin was high, corresponding to up to 35% of the total weight. To this amatoxin vehicle we attached a targeting molecule, human recombinant leucine-21 epidermal growth factor (hrEGFL), via a disulfide-containing linker moiety. A typical average stoichiometry of the hrEGFL labeled toxin conjugate was (L-Orn)164(beta-amanitin)19(COC2H4SSC2H4CO-hrEGFL)2. The affinity for EGF receptors of hrEGFL bound in this conjugate was tested by using A 431 cells. The affinity was eight times lower than that of unsubstituted hrEGFL but regarded as high enough for studying specific toxicity effects with cells bearing EGF receptors. We found that beta-amanitin in the labeled conjugate was able to inhibit the growth of A 431 cells at a concentration of 28 nM, 80 times lower than for native beta-amanitin and 20 times lower than for poly-L-ornithine-bound beta-amanitin without the hrEGFL label. The approximately 20-fold enhancement of cytotoxicity suggests a specific internalization of the toxin conjugate mediated by the hormone label. This idea is supported by the fact that also in another transformed fibroblast cell line, with an increased though smaller number of EGF receptors than A 431 cells, the corresponding enhancement of cytotoxicity was demonstrable but less pronounced (7-fold). The hormone-mediated increase in cytotoxicity of EGF labeled poly-L-ornithine-beta-amanitin conjugates, combined with their moderate toxicity in the mouse, encourages further examination of such compounds in tumor model systems in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 2408344 |
Human alpha 2-macroglobulin gene is located on chromosome 12. |
10.1007/bf01534685 |
Somat. Cell Mol. Genet. |
Human alpha 2-macroglobulin gene is located on chromosome 12.
Abstract
- A cDNA clone encoding amino acids 809-1451 of the protease inhibitor alpha 2-macroglobulin has been isolated from an adult human liver cDNA library. This cDNA was used to examine DNA samples prepared from a panel of human-mouse somatic cell hybrids with different numbers and combinations of human chromosomes for the presence of the human alpha 2-macroglobulin gene. The cosegregation of this gene and chromosome 12 in the cell hybrid panel indicated that the alpha 2-macroglobulin structural gene (designated A2M) is on human chromosome 12.
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| 2409974 |
Bacterial lipopeptides induce ion-conducting pores in planar bilayers |
10.1016/0006-291x(85)91985-0. |
Biochem Biophys Res Commun |
Bacterial lipopeptides induce ion-conducting pores in planar bilayers
Abstract
- Bacterial lipopeptides, known for their antibiotic activities, have been tested for their ability to interact with lipid membranes. These lipopeptides, Iturin A, Bacillomycin L and D and Peptidolipin NA present analogous structural characteristics: a heptapeptidic cycle is linked to a hydrocarbon chain. We present evidence that these lipopeptides modify the conductance of planar bilayers by forming ion-conducting pores.
|
| 2410919 |
Actinomycin and DNA transcription |
10.1073/pnas.82.16.5328 |
Proceedings of the National Academy of Sciences of the United States of America |
Actinomycin and DNA transcription
Abstract
- Recent advances in understanding how actinomycin binds to DNA have suggested its mechanism of action. Actinomycin binds to a premelted DNA conformation present within the transcriptional complex. This immobilizes the complex, interfering with the elongation of growing RNA chains. The model has a number of implications for understanding RNA synthesis.
|
| 2412488 |
Binding and neutralization of bacterial lipopolysaccharide by colistin nonapeptide |
10.1128/AAC.28.1.107. |
Antimicrob Agents Chemother |
Binding and neutralization of bacterial lipopolysaccharide by colistin nonapeptide
Abstract
- Polymyxin nonapeptides, proteolytic derivatives of polymyxin antibiotics, are less toxic than their parent compounds but retain some of their antibacterial activities. To confirm and expand observations that polymyxin nonapeptides have anti-endotoxin activity, we studied the ability of colistin nonapeptide to bind to bacterial lipopolysaccharide (LPS) and to inhibit the effects of LPS on Limulus amoebocyte lysate and lymphocyte mitogenicity. Colistin nonapeptide was purified by high-pressure liquid chromatography and was demonstrated to bind to LPS by equilibrium dialysis. The ability of colistin nonapeptide to render E. coli ATCC 25922 cells sensitive to erythromycin was abrogated by 50% after incubation with E. coli O18 LPS in a ratio by weight of LPS to colistin nonapeptide of 3.9:1. The presence of 4 micrograms of colistin nonapeptide or colistin per ml increased by 130- and 800-fold, respectively, the concentration of E. coli O113 LPS required to produce 50% gelation of Limulus amoebocyte lysate as measured by a spectrophotometric assay. Neutralization of LPS by colistin nonapeptide was time and concentration dependent. In contrast to the neutralization seen with LPS derived from a colistin-sensitive organism, colistin nonapeptide neutralized very little LPS extracted from a strain of Serratia marcescens that was resistant to colistin. Colistin nonapeptide also inhibited LPS-induced 3Hthymidine uptake by splenic lymphocytes, but its activity was less than 1/10 that of colistin. We conclude that colistin nonapeptide binds to LPS and possesses antiendotoxin activity. However, the anti-endotoxin activity of the nonapeptide is considerably less than that of its parent compound, colistin.
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| 2430963 |
Primary structure of human alpha 2-macroglobulin. Complete disulfide bridge assignment and localization of two interchain bridges in the dimeric proteinase binding unit. |
10.1016/s0021-9258(18)66643-8 |
J. Biol. Chem. |
Primary structure of human alpha 2-macroglobulin. Complete disulfide bridge assignment and localization of two interchain bridges in the dimeric proteinase binding unit.
Abstract
- The disulfide bridge pattern of human alpha 2-macroglobulin (alpha 2M) given earlier (Sottrup-Jensen, L., Stepanik, T. M., Kristensen, T., Wierzbicki, D. M., Jones, C. M., Lønblad, P. B., Magnusson, S., and Petersen, T. E. (1984) J. Biol. Chem. 259, 8318-8327) has been revised by showing that Cys255 and Cys408 in one subunit are bridged to Cys408 and Cys255, respectively, in the adjacent subunit of the proteinase binding dimer. Thus, the alpha 2M-dimer contains two interchain disulfide bridges, and the individual subunits are arranged in an antiparallel fashion. These
Results are the outcome of partial reduction experiments, where reduction of methylamine-treated alpha 2M with 1-8 mM mercaptoethanesulfonate at pH 8.0 resulted in the appearance of 2.6 mol of SH-groups per mol of free subunit. Apart from reduction of the two interchain bridges, the intrachain bridges Cys228-Cys276, Cys572-Cys748, Cys798-Cys826, and Cys824-Cys860 are reduced to a minor extent under these conditions. The disulfide bridge pattern of alpha 2M has been completed by showing that the alpha 2M subunit contains 11 intrachain bridges, including a bridge connecting Cys447 with Cys540.
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| 2435749 |
Adsorption of cyclic peptides analogous to gramicidin S and gratisin onto octadecylsilica stationary phase and bacterial cells |
10.1016/0378-4347(87)80241-4. |
J Chromatogr |
Adsorption of cyclic peptides analogous to gramicidin S and gratisin onto octadecylsilica stationary phase and bacterial cells
Abstract
|
| 2437930 |
Determination of interproton distances in peptides by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Conformation of a cyclic analog of substance P in a solution |
None |
Bioorg Khim |
Determination of interproton distances in peptides by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Conformation of a cyclic analog of substance P in a solution
Abstract
- Quantitative method is developed for evaluation interproton distances in peptides in solution. The method is based on the measurement of the relative intensities of the cross-peaks in the pure-phase absorption NOESY spectra. The ratios of the cross-peak intensities IN alpha/I alpha N and INN/I alpha N enable to determine the corresponding interproton distances dN alpha, d alpha N and dNN for several amino acid residues. These distances can be used to estimate other distances with cross-peaks in NOESY spectra. As example, the interproton distances are determined in a cyclic hexapeptide, namely cyclic analogue of substance P: cyclo H-Glu-Phe-Phe-Gly-Leu-Met-NH(CH2)3-NH-. The spatial structure of the molecule in dimethylsulphoxide solution is established.
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| 2443391 |
4,4'-(Z)-dehydrophenylalaninegramicidin S with stabilized bioactive conformation and strong antimicrobial activity |
10.1016/0014-5793(87)80380-0. |
FEBS Lett |
4,4'-(Z)-dehydrophenylalaninegramicidin S with stabilized bioactive conformation and strong antimicrobial activity
Abstract
- Dehydrophenylalanine (delta Phe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D-Phe4,4' to prepare an unsaturated analog. Conformational analysis with 1H-NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val-NH and Leu-CO in the unsaturated analog are strengthened by the incorporation of delta Phe4,4'. This resulted in the reinforcement of the beta-sheet structure which is the most important structural element for GS bioactivity. delta Phe4,4'gramicidin S exhibited indeed very strong antimicrobial activities against Gram-positive bacteria as well as the natural peptide."
|
| 2448768 |
Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes |
10.1073/pnas.85.1.208. |
Proc Natl Acad Sci U S A |
Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes
Abstract
- The B1 (CD20) molecule is a Mr 33,000 phosphoprotein on the surface of human B lymphocytes that may serve a central role in the humoral immune response by regulating B-cell proliferation and differentiation. In this report, a cDNA clone that encodes the B1 molecule was isolated and the amino acid sequence of B1 was determined. B-cell-specific cDNA clones were selected from a human tonsillar cDNA library by differential hybridization with labeled cDNA derived from either size-fractionated B-cell mRNA or size-fractionated T-cell mRNA. Of the 261 cDNA clones isolated, 3 cross-hybridizing cDNA clones were chosen as potential candidates for encoding B1 based on their selective hybridization to RNA from B1-positive cell lines. The longest clone, pB1-21, contained a 2.8-kilobase insert with an 891-base-pair open reading frame that encodes a protein of 33 kDa. mRNA synthesized from the pB1-21 cDNA clone in vitro was translated into a protein of the same apparent molecular weight as B1. Limited proteinase digestion of the pB1-21 translation product and B1 generated peptides of the same sizes, indicating that the pB1-21 cDNA encodes the B1 molecule. Gel blot analysis indicated that pB1-21 hybridized with two mRNA species of 2.8 and 3.4 kilobases only in B1-positive cell lines. The amino acid sequence deduced from the pB1-21 nucleotide sequence apparently lacks a signal sequence and contains three extensive hydrophobic regions. The deduced B1 amino acid sequence shows no significant homology with other known proteins.
|
| 2452834 |
Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors. |
10.1172/jci113478 |
J. Clin. Invest. |
Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.
Abstract
- The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.
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