RESUMEN
BACKGROUND AND OBJECTIVE: Anti-IL-17A IgG/κ monoclonal antibody CJM112 binds both IL-17A and IL-17AF. The purpose of this First-in-Human study was to assess CJM112 effects on safety and efficacy in patients with moderate to severe plaque psoriasis. METHODS: This study had two parts: single ascending doses of 5-450 mg subcutaneous (s.c.) CJM112 (SAD) and multi-dose parallel groups of CJM112 15 mg, 50 mg and 150 mg s.c. low frequency or high frequency (MD). SAD/MD were double-blind, randomized and placebo-controlled; MD also included a secukinumab 150 mg s.c. arm as an active comparator. Patients 18-65 years with moderate to severe psoriasis were included in this study. The efficacy outcome was the change in Psoriasis Area Severity Index (PASI) from baseline to Week 4 in the SAD part of the study, and from baseline to Week 12 in the MD part. RESULTS: 96 patients were enrolled in this study (SAD, n = 42; MD, n = 54). In SAD, CJM112 doses from 15 mg and above demonstrated higher PASI responses compared with placebo at Week 12. CJM112 450 mg did not add further efficacy, but efficacy duration was prolonged compared with CJM112 150 mg. CJM112 MD resulted in a dose-dependent decrease in PASI over time to Week 12. CJM112 150 mg high frequency did not exceed the effect of CJM112 150 mg low frequency and had similar efficacy to secukinumab 150 mg. The safety profile of CJM112 was as expected for an antibody targeting IL-17A/IL-17AF. CONCLUSIONS: CJM112 had clinical efficacy in moderate to severe psoriasis and was generally safe and well tolerated in the doses tested. Additional neutralization of IL-17AF did not translate to increased clinical efficacy compared with secukinumab.
Asunto(s)
Interleucina-17 , Psoriasis , Anticuerpos Monoclonales/efectos adversos , Método Doble Ciego , Humanos , Psoriasis/tratamiento farmacológico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.
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Proteínas Adaptadoras Transductoras de Señales , Lactamas/síntesis química , Fosfopéptidos/síntesis química , Proteínas/química , Dominios Homologos src , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Proteína Adaptadora GRB2 , Lactamas/química , Lactamas/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Ectopic pregnancy is the leading cause of pregnancy-related death during the first trimester. Bilateral ectopic pregnancy is a rare phenomenon, varying in frequency between 1 per 725 and 1 per 1,580 ectopic pregnancies. We report the case of a bilateral ectopic pregnancy (ruptured right cornual and intact left ampullary) in a patient with no known risk factors for extrauterine gestation. CASE: A 33-year-old, black woman, gravida 2, para 1001, presented at approximately 7 weeks' gestation with the acute onset of abdominal pain. She had a rigid surgical abdomen but was hemodynamically stable. Her beta-human chorionic gonadotropin level was 6,398 mIU/mL, and transvaginal ultrasound failed to reveal an intrauterine gestation, adnexal mass or cul-de-sac fluid. Findings at laparotomy included a 500-mL hemoperitoneum and a ruptured right cornual and intact left ampullary pregnancy. Pathology of both specimens confirmed the presence of chorionic villi. CONCLUSION: Although rare, heterotopic pregnancies can occur even in patients without risk factors.
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Embarazo Ectópico/complicaciones , Embarazo Tubario/complicaciones , Adulto , Femenino , Edad Gestacional , Humanos , Embarazo , Embarazo Ectópico/cirugía , Embarazo Tubario/cirugía , Factores de Riesgo , Rotura EspontáneaRESUMEN
A sensitive and specific radioimmunoassay for a novel luteinizing-hormone-releasing-hormone (LHRH) agonist, [2-Me-D-Trp6, DesGly10]LHRH ethylamide (meterelin), was developed for documenting the pharmacokinetic parameters of this peptide following its intravenous (iv) and subcutaneous (sc) administration in dogs. The assay was also used for monitoring meterelin in plasma following its release from d,l-lactide-glycolide implants in the same species. Rabbit antisera generated against [DespyroGlu1] meterelin and conjugated to bovine serum albumin with glutaraldehyde showed high specificity, whereas crossreactivity to LHRH and its fragments and to analogs with substitutions at residues 6 and 10 was found insignificant. The assay was validated in terms of accuracy (recovery range, 94.0-105.4%), in terms of precision (intra- and interassay variations of 10.0-12.4% and 8.6-11.3%, respectively), and in terms of sensitivity (minimum detectable dose of 2.7 pg/assay). Following iv acute administration, a biexponential decline of plasma meterelin levels was observed, with distribution and elimination half-lives of 5.9 +/- 2.5 and 106 +/- 22 min, respectively. After sc acute administration, the elimination half-life was in the range of 103 to 173 min. The systemic clearance (CLT) ranged from 1.6 to 2.6 mL/min/kg, and the volume of distribution at steady state (Vdss) varied from 285 to 438 mL/kg. The elimination half-life (T1/2 beta), Vdss, and ClT were not significantly different after both routes of administration over the 1-100-microgram/kg dose range of peptide studied. Castrate levels of testosterone were attained 10 days after sc administration of the implant, lasted for up to 247 days, and were well correlated with plasma levels of meterelin.
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Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/agonistas , Secuencia de Aminoácidos , Animales , Perros , Implantes de Medicamentos , Hormona Liberadora de Gonadotropina/administración & dosificación , Hormona Liberadora de Gonadotropina/sangre , Hormona Liberadora de Gonadotropina/inmunología , Hormona Liberadora de Gonadotropina/farmacocinética , Sueros Inmunes , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Datos de Secuencia Molecular , Radioinmunoensayo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Testosterona/sangreRESUMEN
The first successful preparation of mono- and disubstituted 3,7-dihydroxytropolone involves a four-step synthetic scheme. Thus, bromination of 3,7-dihydroxytropolone (8) followed by permethylation of the resultant products furnished gram quantities of intermediates 13-18. Single or double Suzuki coupling reactions between these permethylated monobromo- and dibromodihydroxytropolone derivatives and a variety of boronic acids delivered the expected products whose deprotection yielded the desired compounds 1a-u and 26a-n, usually in fair to good yields. Tropolones 1 and 26 were found to be potent inhibitors of inositol monophosphatase with IC50 values in the low-micromolar range. The results are discussed in the context of the recently described novel mode of inhibition of the enzyme by 3,7-dihydroxytropolones.
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Inhibidores Enzimáticos/síntesis química , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Sitios de Unión , Difosfonatos/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Humanos , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Recombinantes/metabolismo , Tropolona/análogos & derivados , Tropolona/síntesis química , Tropolona/metabolismo , Tropolona/farmacologíaRESUMEN
A portion of the ligand binding domain for atrial natriuretic peptide (ANP) was identified as an affinity cross-linked proteolytic fragment of bovine adrenal natriuretic peptide receptor type-A (NPR-A). Affinity purified NPR-A was UV-cross-linked to the amino terminus of 125I-[Tyr2] rat ANP-(2-27). A chymotryptic fragment of the affinity labeled NPR-A was isolated by chromatography and electrophoresis. This fragment yielded a major microsequence corresponding to a region from Met173 to Phe188 of the receptor extracellular domain and containing one N-glycosylation site at Asn180. Bovine NPR-A receptor was then cross-linked to the carboxy terminus of the highly efficient photoaffinity derivative 125I-[Tyr18,Bpa27] rat ANP(1-27). Proteolysis of the affinity labeled NPR-A with cyanogen bromide and trypsin produced radiolabeled and glycosylated fragments of size 15 and 9 kDa, respectively, which contained the epitope Ile181-Phe188 (CS328) and which were detectable by immunoprecipitation with a monospecific polyclonal antibody against CS328. Proteolysis with cyanogen bromide followed by Glu-C produced a shorter photolabeled 6 kDa fragment which was not immunoprecipitable by anti-CS328 antibody and which was not glycosylated. The results lead to the identification of the short segment Asp191-Arg198 as the site of covalent binding of [Tyr18,Bpa27] rat ANP(1-27). This hydrophilic region is adjacent to the epitope Ile181-Phe188 and to the glycosylation site Asn180. It displays the species variability and the high surface probability expected for a portion of the binding domain of NPR-A in contact with ANP.
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Guanilato Ciclasa/química , Natriuréticos/química , Receptores del Factor Natriurético Atrial/química , Marcadores de Afinidad/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Bovinos , Quimotripsina/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida , Glicosilación , Guanilato Ciclasa/metabolismo , Humanos , Ligandos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Datos de Secuencia Molecular , Natriuréticos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Receptores del Factor Natriurético Atrial/metabolismo , Serina Endopeptidasas/metabolismo , Tripsina/metabolismo , Zona GlomerularRESUMEN
The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was completely inactivated by amidination with ethyl acetimidate or reductive methylation with formaldehyde and cyanoborohydride. Activity was retained when the active site was protected with Mg2+, Li+, and D,L-myo-inositol 1-phosphate. Using radiolabeling, peptide mapping, and sequence analysis, Lys-36 was shown to be the protected residue, which is responsible for inactivation. Replacing Lys-36 with glutamine produced a mutant protein, K36Q, with similar affinities for the substrate and the activator Mg2+, but a 50-fold lower turnover number as compared to the wild-type enzyme. Crystallographic studies did not indicate any gross structural changes in the mutant as compared to the native form. Initial velocity data were best described by a rapid equilibrium ordered mechanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but inhibition by lithium was greatly reduced and became noncompetitive. The pH dependence of catalysis and the solvent isotope effect on kcat are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitrophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. All data taken together are consistent with the hypothesis that modifying or replacing the lysine residue in position 36 decreases its polarizing effect on one of the catalytic metal ions and prevents the efficient deprotonation of the metal-bound water nucleophile.
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Lisina/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Secuencia de Bases , Catálisis , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Cartilla de ADN , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Especificidad por SustratoRESUMEN
A procedure for the radioimmunoassay (RIA) of Antarelix (teverelix) in plasma has been developed for the pharmacokinetic study of this potent LHRH antagonist in dogs. Antiserum was produced by coupling the deamidated Antarelix analog to bovine serum albumin by a carbodiimide reaction and immunizing rabbits with the conjugate. The crossreactivity of the antiserum with LHRH, LHRH agonist Metereline and LHRH antagonists tested was negligible, except for Antide which displayed a crossreactivity of 33%. No crossreactivity with Antarelix metabolites could be detected. The RIA is suitable for the direct determination of Antarelix in plasma, with a minimum detectable level of 1.12 fmol/assay. The accuracy and precision of the method were assessed with plasma samples spiked with Antarelix at concentrations ranging from 0.4 to 6.4 pmol/ml. The recovery was 104.8% with intra- and interassay CV between 1 and 3.7%. Pharmacokinetic profiles of Antarelix in dogs were established following an i.v. or a s.c. dose of 10 micrograms/kg.
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Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Antagonistas de Hormonas/sangre , Antagonistas de Hormonas/farmacocinética , Oligopéptidos/sangre , Oligopéptidos/farmacocinética , Animales , Reacciones Cruzadas , Perros , RadioinmunoensayoRESUMEN
The atrial natriuretic R1 receptor is a membrane protein that is present as an apparently preassociated noncovalent oligomer in the absence of ligand as suggested by steric exclusion studies and cross-linking experiments in physiological and recombinant receptor expression systems. The association state of this receptor oligomer was studied in the presence of amiloride and ATP, two known modulators of the R1 receptor functions with both the intact receptor and a cytoplasmic domain-deleted form obtained by limited proteolysis with trypsin. It was shown by steric exclusion on Superose 6 column that amiloride increased the affinity of ANF for the native and truncated receptor, in contrast with ATP, whose destabilizing effect on ANF binding was abolished by truncation of the cytoplasmic domain. Neither amiloride nor ATP exerts its effects by altering the aggregation state of the receptor. Comparison of the measured number of ANF binding sites with immunoassayable receptor protein revealed that the stoichiometry of ANF binding to the R1 receptor was 1:2. This was confirmed by using an ANF analog that bears a photoactivatable group at both of its ends, showing that ANF, as for the growth hormone/receptor complex, interacts with both the receptor subunits and specifically cross-links a dimeric form of the receptor. The potential pharmacological consequences of this 1:2 stoichiometric ratio of the ANF-receptor complex are discussed.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Guanilato Ciclasa/química , Receptores del Factor Natriurético Atrial/química , Zona Glomerular/metabolismo , Adenosina Trifosfato/farmacología , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Reactivos de Enlaces Cruzados , Citoplasma , Guanilato Ciclasa/metabolismo , Técnicas In Vitro , Sustancias Macromoleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Agregación de Receptores/efectos de los fármacos , Receptores del Factor Natriurético Atrial/metabolismo , Relación Estructura-ActividadRESUMEN
Interleukin-1 (IL-1) molecules are cytokines involved in the acute-phase response against infection and injury. Three naturally occurring IL-1 molecules are known, two agonists: IL-1 alpha and IL-1 beta, and one antagonist, the IL-1 receptor antagonist (IL-1ra). Although IL-1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL-1ra has been solved at 0.21-nm resolution by molecular replacement using the IL-1 beta structure as a search model. The crystals contain two independent IL-1ra molecules which are very similar. IL-1ra has the same fold as IL-1 alpha and IL-1 beta. The fold consists of twelve beta-strands which form a six-stranded beta-barrel, closed on one side by three beta-hairpin loops. Cys69 and Cys116 are linked via a disulfide bond and Pro53 has been built in the cis-conformation. Comparison of the IL-1ra structure with the IL-1 alpha and IL-1 beta structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N-terminus up to the beginning of strand beta 1 and the loops D and G, is very different in the three IL-1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL-1 receptor.
Asunto(s)
Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/química , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Disulfuros/química , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/química , Interleucina-1/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Prolina/química , Conformación Proteica , Homología de Secuencia de Aminoácido , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , TermodinámicaRESUMEN
BACKGROUND: Aminopeptidases specifically cleave the amino-terminal residue from polypeptide chains and are involved in the metabolism of biologically active peptides. The family includes zinc-dependent enzymes possessing either one or two zinc ions per active site. Structural studies providing a detailed view of the metal environment may reveal whether the one-zinc and two-zinc enzymes constitute structurally and mechanistically distinct subclasses, and what role the metal ions play in the catalytic process. RESULTS: We have solved the crystal structure of the monomeric aminopeptidase from Aeromonas proteolytica at 1.8 A resolution. The protein is folded into a single alpha/beta globular domain. The active site contains two zinc ions (3.5 A apart) with shared ligands and symmetrical coordination spheres. We have compared it with the related bovine lens leucine aminopeptidase and the cobalt-containing Escherichia coli methionine aminopeptidase. CONCLUSIONS: The environment and coordination of the two zinc ions in A. proteolytica aminopeptidase strongly support the view that the two metal ions constitute a co-catalytic unit and play equivalent roles during catalysis. This conflicts with the conclusions drawn from the related bovine leucine aminopeptidase and early biochemical studies. In addition, the known specificity of the aminopeptidase for hydrophobic amino-terminal residues is reflected in the hydrophobicity of the active site cleft.
Asunto(s)
Aeromonas/enzimología , Aminopeptidasas/química , Proteínas Bacterianas/química , Conformación Proteica , Sitio Alostérico , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Proteínas del Ojo/química , Leucil Aminopeptidasa/química , Modelos Moleculares , ZincRESUMEN
The structure of aldose reductase, a monomeric enzyme of 314 amino acids which crystallizes in space group P1 with four monomers per asymmetric unit, has been solved using a combination of single isomorphous replacement (SIR), solvent flattening and local symmetry averaging. The self rotation showed evidence of 222 local symmetry. The map calculated from the original single isomorphous replacement phases showed a clear solvent envelope but was uninterpretable. A first averaging attempt failed because the molecular envelope obtained from the SIR map weighted with monomer correlation was too small and the averaging was biased by low-resolution truncation. A second attempt with an enlarged envelope and including low-resolution reflections succeeded in refining phases at 3.5 A resolution but failed to extend them correctly. Rigid-body refinement of a partial model based on the 3.5 A map calculated from refined phases showed significant departures from the 222 symmetry. A third averaging attempt using the improved symmetry succeeded in producing a clear map with phases extended to 3.07 A resolution. This map revealed a (beta/alpha)(8) fold, not previously found in NADPH-dependent enzymes. This work shows the importance of mask definition and local symmetry elements accuracy for averaging, and describes a method for improving these parameters.
RESUMEN
Poison Centers frequently rely on the assistance of local plant nurseries to identify unknown plants involved in exposures. The reliability and accuracy of utilizing this method has never been studied; therefore, our objective was to evaluate this primary resource of plant identification. A study was done in which callers were instructed to take plant samples to a local nursery for visual identification. Once the patient was treated according to our normal protocol, the plant specimen was sent to a botanist for a second identification. The botanist provided his identification results through a blinded process. The collected data was gathered from 68 cases that completed the necessary study criteria. In 58% of the cases, plant nurseries were an unreliable source for plant identification. These incorrect identifications resulted in the "undertreatment" in 24% of the exposures.
Asunto(s)
Intoxicación por Plantas , Plantas Tóxicas , Animales , HumanosRESUMEN
The 130 kDa atrial natriuretic factor receptor (ANF-R1) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99-126) and there is no detectable intrinsic kinase activity associated with the ANF-R1 receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for 125I-ANF. However, we have demonstrated a significant 32P incorporation in the ANF-R1 receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R1 receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.
Asunto(s)
Naftalenos , Proteína Quinasa C/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Secuencia de Aminoácidos , Animales , Factor Natriurético Atrial/antagonistas & inhibidores , Factor Natriurético Atrial/metabolismo , Unión Competitiva , Bovinos , GMP Cíclico/metabolismo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Ésteres del Forbol/farmacología , Fosforilación , Compuestos Policíclicos/farmacología , Receptores del Factor Natriurético Atrial/efectos de los fármacos , Zona Glomerular/metabolismoRESUMEN
Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Cristalino/química , Aldehído Reductasa/química , Animales , Espectrometría de Masas/métodos , PorcinosRESUMEN
Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.
Asunto(s)
Aldehído Reductasa/química , NADP/metabolismo , Aldehído Reductasa/metabolismo , Sitios de Unión , Sustancias Macromoleculares , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Difracción de Rayos X/métodosRESUMEN
A polyclonal antibody monospecific for an intracellular epitope of the atrial natriuretic factor (ANF)-R1 receptor was produced. The receptor protein (200 pmoles) was purified to homogeneity from bovine adrenal zona glomerulosa (BAZG), reduced, alkylated and digested with trypsin. The tryptic fragments were purified by reverse-phase h.p.l.c. on a C18 column. Based on the sequence of one of these fragments, a peptide was chemically synthesized, coupled to thyroglobulin and injected into rabbits. The antibody obtained was shown to be specific for the R1-type as no receptor was detected in bovine red blood cells (RBC) (which are devoid of ANF receptors) and in NIH-3T3 cell membranes (where only the R2-type is expressed). Several other tissues were screened and comparison of the immunoreactive receptor density estimates with those obtained by ANF binding yielded a correlation coefficient (r2) of 0.965. The minimal detectable dose was typically 3 fmoles/tube and the ED50 of the RIA was 30 fmoles/tube. Cyanogen bromide digestion of the receptor was essential for antigenic detection, indicating that the epitope is probably hindered due to the tertiary structure of the native protein. Moreover, location of the epitope in the kinase homology domain of the receptor, combined with partial tryptic digestion, suggests that the proteolysis-sensitive region of the receptor is located between the transmembrane-spanning domain and the amino acid 586. This method of production of antibodies should be useful to precisely map the amino acids involved in various functions of the receptor.
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Glándulas Suprarrenales/inmunología , Formación de Anticuerpos/inmunología , Receptores del Factor Natriurético Atrial/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Factor Natriurético Atrial/metabolismo , Bovinos , Epítopos/inmunología , Guanilato Ciclasa/metabolismo , Radioisótopos de Yodo , Datos de Secuencia Molecular , Proteínas Quinasas/química , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , TripsinaAsunto(s)
Aldehído Reductasa/química , Cristalino/enzimología , Aldehído Reductasa/aislamiento & purificación , Aldehído Reductasa/metabolismo , Animales , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Indicadores y Reactivos , Cinética , Sustancias Macromoleculares , Espectrometría de Masas , Peso Molecular , PorcinosRESUMEN
The bovine adrenal angiotensin II receptor was solubilized with the non-ionic detergent octyl beta-D-glucoside following its binding with the high-affinity antagonist 125I-labelled [Sar1,Ile8]angiotensin II. The complex was sufficiently stable to allow the determination of its hydrodynamic properties. The solubilized receptor migrated on a Superose 6 column as a single peak with a Stokes radius of 5.29 nm. Comparison of sedimentation behaviour through a sucrose density gradient in H2O and 2H2O lead to a partial specific volume of 0.751 ml/g and a sedimentation coefficient (S20,w) of 5.17 S. Combination of gel filtration and sedimentation data indicated that the labelled protein-detergent complex has an Mr of 124,000 and a frictional ratio of 1.42. The Mr of the angiotensin II receptor was estimated as 104,000 kDa after correction for the bound detergent. Photoaffinity cross-linking of 125I-[Sar1, (4-N3)Phe8]angiotension II with bovine adrenal membrane receptor followed by SDS/PAGE under reducing and non-reducing conditions yielded a broad band of Mr 54,000. This suggests that the angiotensin II receptor is a non-covalent dimer in which the two subunits have a similar Mr.
Asunto(s)
Angiotensina II/metabolismo , Receptores de Angiotensina/metabolismo , Zona Glomerular/metabolismo , Marcadores de Afinidad , Angiotensina II/análogos & derivados , Animales , Unión Competitiva , Bovinos , Detergentes , Glucósidos , Solubilidad , AguaRESUMEN
In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.