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1.
J Biol Chem ; 270(46): 27905-13, 1995 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-7499265

RESUMO

The ST2/T1 receptor, a homologue of the interleukin 1 receptor (IL-1R), was expressed in COS and Drosophila S2 cells as a human IgG-Fc fusion protein. While a type I IL-1RFc fusion protein bound human IL-1 in vitro, the ST2Fc fusion protein did not. Furthermore, IL-1 stimulated a synthetic interleukin-8 promoter reporter gene that was cotransfected into Jurkat cells with a full-length IL-1R type I (IL-1RI) or a chimeric receptor composed of the IL-1RI extracellular domain and ST2 intracellular domain. In contrast, IL-1 did not stimulate the interleukin-8 promoter when cotransfected with a full-length ST2 or an ST2 extracellular/IL-1R intracellular domain fusion protein. Both IL-1RI and the IL-1R/ST2R chimeric receptor also activated a receptor-associated kinase and CSBP/p38 MAP kinase. Using ST2Fc receptor, we have identified, through receptor precipitation, receptor-dot blot and surface plasmon resonance, a putative ligand of ST2 secreted from Balb/c 3T3 and human umbilical vein endothelial cells. The putative ligand was also able to stimulate CSBP/p38 MAP kinase through the ST2 receptor. These results suggest that the ST2 is not an IL-1 receptor but rather has its own cognate ligand.


Assuntos
Endotélio Vascular/metabolismo , Interleucina-1/farmacologia , Proteínas de Membrana , Proteínas/metabolismo , Receptores de Interleucina-1/metabolismo , Células 3T3 , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA , Drosophila melanogaster , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Interleucina-1/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-8/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Receptores de Superfície Celular , Receptores de Interleucina , Receptores de Interleucina-1/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Transfecção , Veias Umbilicais
2.
J Recept Res ; 14(6-8): 357-79, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7877135

RESUMO

The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.


Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/genética , Adenilil Ciclases/genética , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Cobre/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Regiões Promotoras Genéticas , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , Transducina/farmacologia
3.
Biochemistry ; 32(48): 13054-60, 1993 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-8241159

RESUMO

Simian immunodeficiency virus (SIV) proteins have considerable amino acid sequence homology to those from human immunodeficiency virus (HIV); thus monkeys are considered useful models for the preclinical evaluation of acquired immune deficiency syndrome (AIDS) therapeutics. We have crystallized and determined the three-dimensional structure of SIV protease bound to the hydroxyethylene isostere inhibitor SKF107457. Crystals of the complex were grown from 25-32% saturated sodium chloride, by the hanging drop method of vapor diffusion. They belong to the orthorhombic space group I222, with a = 46.3 A, b = 101.5 A, and c = 118.8 A. The structure has been determined at 2.5-A resolution by molecular replacement and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of - magnitude of Fc parallel/sigma magnitude of Fo magnitude of), of 0.189. The overall structure of the complex is very similar to previously reported structures of HIV-1 protease bound to inhibitors. The inhibitor is bound in a conformation that is almost identical to that found for the same inhibitor bound to HIV-1 protease, except for an overall translation of the inhibitor, varying along the backbone atoms from about 1.0 A at the termini to about 0.5 A around the scissile bond surrogate. The structures of the SIV and HIV-1 proteins vary significantly only in three surface loops composed of amino acids 15-20, 34-45, and 65-70. Superposition of the 1188 protein backbone atoms from the two structures gives an rms deviation of 1.0 A; this number is reduced to 0.6 A when atoms from the three surface loops are eliminated from the rms calculation.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Ácido Aspártico Endopeptidases/ultraestrutura , Vírus da Imunodeficiência Símia/enzimologia , Sequência de Aminoácidos , Antivirais/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Cristalografia por Raios X , Inibidores da Protease de HIV/química , Ligação de Hidrogênio , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
4.
Biochemistry ; 32(31): 7972-80, 1993 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-8347601

RESUMO

The human immunodeficiency virus type 1 (HIV-1) protease is a potential target of acquired immune deficiency syndrome (AIDS) therapy. A highly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV-1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has been determined at 2.3-A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel F(o) magnitude to - Fc parallel/sigma magnitude of F(o)), of 0.178. The inhibitor is held in the enzyme active site by a set of hydrophobic and hydrophilic interactions, including an interaction between Arg8 and the center of the terminal benzene rings of the inhibitor. The phosphinate establishes a novel interaction with the two catalytic aspartates; each oxygen of the central phosphinic acid moiety interacts with a single oxygen of one aspartic acid, establishing a very short (2.2-2.4 A) oxygen-oxygen contact. As with the structures of penicillopepsin bound to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka, N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochemistry 31, 5201-14], we interpret this short distance and the stereochemical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proton located midway between the two atoms.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Inibidores da Protease de HIV/síntese química , HIV-1/enzimologia , Compostos Organofosforados/síntese química , Ácidos Fosfínicos , Valina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Catálise/efeitos dos fármacos , Cristalização , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Álcoois Açúcares/química , Valina/síntese química , Valina/química , Valina/metabolismo , Difração de Raios X
5.
J Biol Chem ; 267(32): 22770-8, 1992 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-1429626

RESUMO

As part of a structure-based drug design program directed against enzyme targets in the human immunodeficiency virus (HIV), we have determined the three-dimensional structures of the HIV type 1 protease complexed with two hydroxyethylene-based inhibitors. The inhibitors (SKF 107457 and SKF 108738) are hexapeptide substrate analogues with the scissile bond being replaced by a hydroxyethylene isostere. The structures were determined using x-ray diffraction data to 2.2 A measured at the Cornell High Energy Synchrotron Source on hexagonal crystals of each of the complexes. The structures have been extensively refined using a reciprocal space least-squares method to conventional crystallographic R factors of 0.186 and 0.159, respectively. The protein structure differs from that in the unliganded state of the enzyme and is most similar to that of the structure of the other reported (Jaskolski, M., Tomasselli, A. G., Sawyer, T. K., Staples, D. G., Heinrikson, R. L., Schneider, J., Kent, S. B. H., and Wlodawer, A. (1990) Biochemistry 29, 5889-5907) hydroxyethylene-based inhibitor complex. Unlike in that structure, however, the inhibitors are observed, in the present crystal structures, in two equally abundant orientations that are a consequence of the homodimeric nature of the enzyme coupled with the asymmetric structures of the inhibitors. Although the differences between the two inhibitors used in the present study are confined to the P1' site, the van der Waals interactions made by the inhibitor atoms with the amino acid residues in the protein differ throughout the structures of the inhibitors.


Assuntos
Etilenos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Protease de HIV/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Protease de HIV/química , HIV-1/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Conformação Proteica , Relação Estrutura-Atividade , Difração de Raios X/métodos
6.
Biochemistry ; 31(39): 9491-501, 1992 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-1390732

RESUMO

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0


Assuntos
Ácido Aspártico Endopeptidases/química , Protease de HIV/química , HIV-1/enzimologia , Vírus da Imunodeficiência Símia/enzimologia , Ácido Aspártico Endopeptidases/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Protease de HIV/efeitos dos fármacos , Temperatura Alta , Modelos Químicos , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Termodinâmica , Ultracentrifugação , Ureia/farmacologia
7.
Biochemistry ; 30(34): 8424-34, 1991 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-1883829

RESUMO

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.


Assuntos
Endopeptidases/isolamento & purificação , Protease de HIV/isolamento & purificação , HIV-1/enzimologia , Proteínas dos Retroviridae/isolamento & purificação , Vírus da Imunodeficiência Símia/enzimologia , Sequência de Aminoácidos , Animais , Endopeptidases/química , Endopeptidases/classificação , Protease de HIV/classificação , Protease de HIV/genética , Inibidores da Protease de HIV , HIV-1/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Conformação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/classificação , Proteínas Recombinantes/isolamento & purificação , Proteínas dos Retroviridae/antagonistas & inibidores , Proteínas dos Retroviridae/classificação , Proteínas dos Retroviridae/genética , Vírus da Imunodeficiência Símia/fisiologia , Especificidade por Substrato
8.
Biochemistry ; 30(34): 8441-53, 1991 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-1883830

RESUMO

The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.


Assuntos
Protease de HIV/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Oligopeptídeos/química , Isótopos de Oxigênio
9.
J Chromatogr ; 435(1): 185-92, 1988 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-3350891

RESUMO

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.


Assuntos
Miocárdio/análise , Miosinas/isolamento & purificação , Aminoácidos/análise , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Cobaias , Salicilatos , Dodecilsulfato de Sódio , Espectrofotometria Ultravioleta
10.
Mol Pharmacol ; 32(1): 195-200, 1987 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3039336

RESUMO

Turkey red blood cell, beta 1-adrenergic receptors (BARs) were prepared to electrophoretic homogeneity by affinity chromatography, size exclusion high performance liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare rabbit polyclonal anti-BAR antibodies. Anti-BAR activity was confirmed by immunoadsorption of [125I]cyanopindolol-labeled BAR to a protein A affinity column using the anti-BAR antibodies. BAR was compared to the catecholamine biosynthetic enzyme dopamine B-hydroxylase (DBH) by anti-BAR antibody cross-reactivity. DBH was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion, and concanavalin A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 units/mg of protein. Homogeneity was confirmed by nondenaturing polyacrylamide gel electrophoresis. Both DBH and BAR were recognized by the anti-BAR antibodies on Western transfer and immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH, suggesting that the anti-BAR antibodies recognize specific portions of DBH amino acid sequence and not associated carbohydrate. DBH-cross-reactive antibodies were also purified by affinity chromatography using immobilized DBH and shown to immunoadsorb [125I]cyanopindolol-labeled BAR by protein A affinity chromatography. These results suggest that the catecholamine biosynthetic enzyme DBH and BAR may be related in structure.


Assuntos
Dopamina beta-Hidroxilase/imunologia , Receptores Adrenérgicos beta/imunologia , Animais , Bovinos , Membrana Celular/imunologia , Cromatografia de Afinidade , Reações Cruzadas , Dopamina beta-Hidroxilase/isolamento & purificação , Glicosilação , Técnicas de Imunoadsorção , Peso Molecular , Receptores Adrenérgicos beta/isolamento & purificação , Perus
11.
Biochem Pharmacol ; 35(21): 3821-5, 1986 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-2877672

RESUMO

Beta 1- and beta 2-adrenergic receptor radioligand antagonist binding activities in plasma membrane preparations from mammalian lung, as well as amphibian and avian red blood cells, have been shown to be inactivated by agents which specifically alkylate tyrosine. In membrane preparations, protection against inactivation was afforded by both agonists and antagonists. In soluble purified preparations, antagonists but not agonists protected against inactivation. These results suggest that tyrosine is located at or near the ligand binding site of both beta 1- and beta 2-adrenergic receptors.


Assuntos
Receptores Adrenérgicos beta/análise , Tirosina/análise , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anuros , Sítios de Ligação/efeitos dos fármacos , Bovinos , Membrana Celular/metabolismo , Cobaias , Ligantes/metabolismo , Pindolol/análogos & derivados , Pindolol/metabolismo , Coelhos , Ratos , Receptores Adrenérgicos beta/metabolismo , Especificidade da Espécie , Perus
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