RESUMEN
Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.
Asunto(s)
Autoanticuerpos/inmunología , Epítopos/inmunología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Ingeniería de Proteínas , Diabetes Mellitus Tipo 1/inmunología , HumanosRESUMEN
An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mutación , Proteínas de Neoplasias , Triptófano/metabolismo , Naftalenosulfonatos de Anilina/farmacología , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes/farmacología , Guanidina/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/química , Espectrofotometría , Espectrofotometría Ultravioleta , Triptófano/química , Rayos UltravioletaRESUMEN
The convenience of combining the measurement of antibodies to glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type 1 and 115 adult-onset diabetic patients. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type 1 diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or within the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence showed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and IA-2A is a useful strategy for prospective screening of type 1 diabetes. However, in adults, the profile of individual markers discloses the course to insulin dependence. Therefore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.
Asunto(s)
Autoanticuerpos/sangre , Diabetes Mellitus Tipo 2/inmunología , Glutamato Descarboxilasa/inmunología , Insulina/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Adolescente , Adulto , Argentina , Biomarcadores , Niño , Diabetes Mellitus Tipo 1/clasificación , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/clasificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Ensayo de Unión RadioliganteRESUMEN
Solvent-induced directional aggregation of human carbonic anhydrase II (hCA) was studied by small angle X-ray scattering and fluorescence and fourth-derivative ultraviolet absorption spectroscopy. We propose that hCA at 5 mg ml(-1) in pure water forms head-to-tail oligomers built up, on average, by four to five monomers. At higher protein concentrations, the oligomers associate pair-wise and side-by-side. Spectroscopic evidence suggests that the subunits forming the aggregates are tightly folded, but with a structure that differs, at least locally, from the native state. A more complex aggregation pattern was observed under solvent conditions that favor the removal of zinc from the enzyme-active site, conditions under which the subunits are significantly less compact than in water. hCA may provide a useful model to investigate the effects of additives and genetic manipulation on protein aggregation.
Asunto(s)
Anhidrasas Carbónicas/química , Dispersión de Radiación , Sitios de Unión , Escherichia coli/química , Humanos , Concentración de Iones de Hidrógeno , Pliegue de Proteína , Espectrometría de Fluorescencia , Espectrometría por Rayos X , Espectrofotometría Ultravioleta , Rayos Ultravioleta , Agua/química , Rayos X , Zinc/químicaRESUMEN
Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.
Asunto(s)
Bacillus/enzimología , Escherichia coli/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Cromatografía/métodos , Escherichia coli/genética , Fluorescencia , Cinética , Datos de Secuencia Molecular , Presión Osmótica , Periplasma/metabolismo , Regiones Promotoras Genéticas , Desnaturalización Proteica , Pliegue de Proteína , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares , beta-Lactamasas/genéticaRESUMEN
Most insulin-dependent diabetes mellitus patients gen-erate conformational autoantibodies to the islet-cell 65-kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type-1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild-type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350-359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino-acid substitution: Met-161-->Thr. This change impeded the co-expression of a 48-kDa by-product from an internal translation site. Also, a second 58-kDa by-product was identified as a GAD65 C-terminal proteolytic fragment that co-purifies with thioredoxin-M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild-type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.
Asunto(s)
Escherichia coli/genética , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Metionina , Datos de Secuencia Molecular , Ingeniería de Proteínas/métodos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , TreoninaRESUMEN
The last three C-terminal residues (129-131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6-->Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129-131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.
Asunto(s)
Ácidos Grasos/metabolismo , Ingeniería de Proteínas , Proteínas/química , Proteínas/genética , Sustitución de Aminoácidos , Animales , Sitios de Unión , Mucosa Intestinal/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/diagnóstico , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Glutamato Descarboxilasa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Adolescente , Enfermedades Autoinmunes/inmunología , Biotina/metabolismo , Niño , Preescolar , Diabetes Mellitus Tipo 1/diagnóstico , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Lactante , Masculino , Radioinmunoensayo/métodos , Sensibilidad y Especificidad , Tiorredoxinas/inmunologíaRESUMEN
By means of a facile chemical modification of the bovine serum albumin molecule, it was possible to measure its hydrodynamic radius with high accuracy (approximately 3%) using the TDPAC technique. The new approach presented here allows a wide use of the TDPAC technique to perform high precision studies of backbone dynamics of almost any protein.
Asunto(s)
Cisteína/química , Ácido Edético/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Fenómenos Químicos , Química Física , Cisteína/metabolismo , Ácido Edético/metabolismo , Radioisótopos de Indio , Conformación Molecular , Albúmina Sérica Bovina/metabolismo , Análisis Espectral/métodos , Factores de TiempoRESUMEN
Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.
Asunto(s)
Glutamato Descarboxilasa/genética , Proteínas Recombinantes de Fusión/genética , Autoanticuerpos/inmunología , Western Blotting , Niño , Clonación Molecular , Diabetes Mellitus Tipo 1/inmunología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Glutamato Descarboxilasa/inmunología , Glutamato Descarboxilasa/metabolismo , Humanos , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Tiorredoxinas/genéticaRESUMEN
The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.
Asunto(s)
Autoanticuerpos/análisis , Glutamato Descarboxilasa/inmunología , Adolescente , Animales , Células CHO , Niño , Preescolar , Cricetinae , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lactante , Masculino , Pruebas de Precipitina , Ratas , Ratas Wistar , Proteínas Recombinantes/inmunologíaRESUMEN
A low-molecular-mass fatty acid-binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica. Purification was achieved by a two-step procedure involving size-exclusion and cation-exchange chromatography. The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N-terminus. Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid-binding proteins.
Asunto(s)
Proteínas Portadoras/química , Proteínas Fúngicas/química , Proteína P2 de Mielina/química , Proteínas de Neoplasias , Saccharomycetales/química , Secuencia de Aminoácidos , Proteínas Portadoras/aislamiento & purificación , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Bromuro de Cianógeno/metabolismo , Citoplasma/química , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión a Ácidos Grasos , Proteínas Fúngicas/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Proteína P2 de Mielina/aislamiento & purificación , Ácido Palmítico/metabolismo , Fragmentos de Péptidos/química , Análisis de Secuencia , Tripsina/metabolismoRESUMEN
High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.
Asunto(s)
Ácidos Palmíticos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Catalasa/metabolismo , Medios de Cultivo , Ácidos Grasos/metabolismo , Microcuerpos/efectos de los fármacos , Microcuerpos/metabolismo , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Análisis de Varianza , Animales , Encéfalo/crecimiento & desarrollo , Proteínas Portadoras/aislamiento & purificación , Cromatografía en Gel , Citosol/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/metabolismo , Femenino , Especificidad de Órganos , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Ratas , Ratas WistarRESUMEN
1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Neoplasias , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Cromatografía en Gel , Proteínas de Unión a Ácidos Grasos , CinéticaRESUMEN
The effect of acetylation of tyrosine residues on the binding capacity of human growth hormone (hGH) to rat liver lactogenic and somatogenic receptors was studied. When 3.7 tyrosine and 4.8 lysine residues were acetylated with N-acetylimidazole, both the in vivo and the in vitro capacities of hGH to compete with 125I-labeled bovine growth hormone for somatogenic binding sites greatly decreased. Acetylation also affected the in vitro binding capacity to lactogenic sites. Most of the somatogenic binding activity was recovered by hydroxylamine treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The same treatment partially restored lactogenic binding capacity. The reactivity of hGH tyrosine residues to N-acetylimidazole, together with previous evidence, suggests that: (a) Tyrosine residues 160 and 164, when acetylated, are likely to be responsible for the low binding activity of acetylated hGH. (b) Tyrosine 160 may play a significant role in hGH interaction with lactogenic receptors.
Asunto(s)
Hormona del Crecimiento/metabolismo , Imidazoles/química , Hígado/química , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos , Receptores de Somatotropina/metabolismo , Tirosina/química , Acetilación , Animales , Sitios de Unión , Cromatografía Líquida de Alta Presión , Femenino , Hormona del Crecimiento/química , Hígado/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Oxidación-Reducción , Ratas , Espectrofotometría UltravioletaRESUMEN
The secondary structure of 11 mammalian growth hormones has been predicted by combining five different methods. Three long helical regions located around residues 20, 120, and 170 constitute the most prominent common feature in the species studied. The strong amphiphilic character of these helices suggests that they can play an important role in protein folding or stability.
Asunto(s)
Hormona del Crecimiento , Animales , Humanos , Mamíferos , Estructura Molecular , Conformación ProteicaRESUMEN
Derivatives of bovine growth hormone, containing monoaminotyrosyl residues in positions 35, 42 and 174, were treated at pH 3.6 with a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Under these conditions aminotyrosyl groups reacted. On changing the pH to 9.3, the second fluorine atom of the reagent was substituted with the sterically adjacent side groups of lysine, since the excess of reagent had been previously removed. The modified protein underwent cyanogen bromide treatment. Peptides containing the crosslinks were purified from tryptic digests of the cyanogen bromide fragments by HPLC. Results show that aminoTyr 174 was able to form dinitrophenylene bridges with Lys 111, Lys 29 and Lys 170. AminoTry 35 was found crosslinked to Lys 29. Taking into account the size of the reagent, it may be inferred that Lys 29, 111 and 170 are located at approximately 5 A from Tyr 174 in the bovine growth hormone molecule.