RESUMO
Encoding information into memory is sensitive to distraction while retrieving that memory may be compromised by proactive interference from pre-existing memories. These two debilitating effects are common in neuropsychiatric conditions, but modelling them preclinically to date is slow as it requires prolonged operant training. A step change would be the validation of functionally equivalent but fast, simple, high-throughput tasks based on spontaneous behaviour. Here, we show that spontaneous object preference testing meets these requirements in the subchronic phencyclidine rat model for cognitive impairments associated with schizophrenia. Subchronic phencyclidine rats show clear memory sensitivity to distraction in the standard novel object recognition task. However, due to this, standard novel object recognition task cannot assess proactive interference. Therefore, we compared subchronic phencyclidine performance in standard novel object recognition task to that using the continuous novel object recognition task, which offers minimal distraction, allowing disease-relevant memory deficits to be assessed directly. We first determined that subchronic phencyclidine treatment did not affect whisker movements during object exploration. Subchronic phencyclidine rats exhibited the expected distraction standard novel object recognition task effect but had intact performance on the first continuous novel object recognition task trial, effectively dissociating distraction using two novel object recognition task variants. In remaining continuous novel object recognition task trials, the cumulative discrimination index for subchronic phencyclidine rats was above chance throughout, but, importantly, their detection of object novelty was increasingly impaired relative to controls. We attribute this effect to the accumulation of proactive interference. This is the first demonstration that increased sensitivity to distraction and proactive interference, both key cognitive impairments in schizophrenia, can be dissociated in the subchronic phencyclidine rat using two variants of the same fast, simple, spontaneous object memory paradigm.
Assuntos
Quimiocina CXCL10/análise , Doença Enxerto-Hospedeiro/diagnóstico , Pele/patologia , Doença Aguda , Adolescente , Estudos de Casos e Controles , Movimento Celular , Criança , Pré-Escolar , Citocinas/análise , Humanos , Lactente , Ativação Linfocitária , Testes do Emplastro , Pele/química , Manejo de Espécimes/métodos , Linfócitos TRESUMO
The skin has an amazing array of complex interacting biological processes. Recent advances in investigational techniques now allow evaluation of these processes at the level of the gene, protein and metabolite. Sometimes collectively known as the omics, these fields of inquiry, known as genomics, proteomics and metabolomics, respectively, are yielding new and important insights into skin structure and processes, its responses to injury and age, and the mechanisms by which new interventions and compounds may work to improve the health and integrity of this crucial organ.
Assuntos
Genômica/tendências , Metabolômica/tendências , Fenômenos Fisiológicos da Pele/genética , Cromatografia/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Previsões , Expressão Gênica/genética , Genômica/métodos , Humanos , Metabolômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteômica/métodos , Proteômica/tendências , Análise Espectral/métodosRESUMO
The aim of the study was to investigate whether the protein and folic acid content of the maternal diet and the sex of the offspring alter the polyunsaturated fatty acid content of hepatic phospholipids and triacylglycerol (TAG). Pregnant rats were fed diets containing 18% or 9% protein with either 1 or 5mg/kg folic acid. Maternal diet did not alter hepatic lipid composition in the adult offspring. Data from each maternal dietary group were combined and reanalysed. The proportion of 18:0, 20:4n-6 and 22:6n-3 in liver phospholipids was higher in females than in males, while hepatic TAG composition did not differ between sexes. Delta5 Desaturase expression was higher in females than in males. Neither Delta5 nor Delta6 desaturase expression was related to polyunsaturated fatty acid concentrations. These results suggest that sex differences in liver phospholipid fatty acid composition may reflect primary differences in the specificity of phospholipid biosynthesis.
Assuntos
Ácidos Graxos Insaturados/análise , Fígado/química , Fosfolipídeos/química , Caracteres Sexuais , Triglicerídeos/análise , Animais , Proteínas Alimentares/administração & dosagem , Ácidos Graxos Dessaturases/metabolismo , Feminino , Ácido Fólico/administração & dosagem , Linoleoil-CoA Desaturase/metabolismo , Fígado/enzimologia , Masculino , Gravidez , RatosRESUMO
The trend in proteomics is to work with increasingly complex protein mixtures, limiting the protein separation steps prior to analysis. This is due in part to the difficulties encountered with detecting low abundance proteins, protein losses during SDS PAGE, and the limited separation capability of even 2D PAGE where a single protein spot may still contain multiple proteins. Hence, the ABRF-PRG02 sample was designed to study a simple protein mixture of five proteins at the approximately 2 pmol and approximately 200 fmol levels. The sample, after a tryptic digestion, was sent out by the Proteomics Research Group of the ABRF to interested member labs. A total of 41 labs participated in this study, with each participant using some type of mass spectrometric analysis. Laboratories that used microLC-NSI (microLC with nanospray ionization) with MS/MS analysis had a higher percent accuracy than labs using MALDI-MS (matrix assisted laser desorption ionization mass spectrometry).
RESUMO
BACKGROUND: Several methods have been developed for creating Cys2His2 zinc finger proteins that recognize novel DNA sequences, and these proteins may have important applications in biological research and gene therapy. In spite of this progress with design/selection methodology, fundamental questions remain about the principles that govern DNA recognition. One hypothesis suggests that recognition can be described by a simple set of rules--essentially a "recognition code"--but careful assessment of this proposal has been difficult because there have been few structural studies of selected zinc finger proteins. RESULTS: We report the high-resolution cocrystal structures of two zinc finger proteins that had been selected (as variants of Zif268) to recognize a eukaryotic TATA box sequence. The overall docking arrangement of the fingers within the major groove of the DNA is similar to that observed in the Zif268 complex. Nevertheless, comparison of Zif268 and the selected variants reveal significant differences in the pattern of side chain-base interactions. The new structures also reveal side chain-side chain interactions (both within and between fingers) that are important in stabilizing the protein-DNA interface and appear to play substantial roles in recognition. CONCLUSIONS: These new structures highlight the surprising complexity of zinc finger-DNA interactions. The diversity of interactions observed at the protein-DNA interface, which is especially striking for proteins that were all derived from Zif268, challenges fundamental concepts about zinc finger-DNA recognition and underscores the difficulty in developing any meaningful recognition code.
Assuntos
Cisteína/química , Histidina/química , TATA Box , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Cys2His2 zinc finger proteins offer a stable and versatile framework for the design of proteins that recognize desired target sites on double-stranded DNA. Individual fingers from these proteins have a simple beta beta alpha structure that folds around a central zinc ion, and tandem sets of fingers can contact neighboring subsites of 3-4 base pairs along the major groove of the DNA. Although there is no simple, general code for zinc finger-DNA recognition, selection strategies have been developed that allow these proteins to be targeted to almost any desired site on double-stranded DNA. The affinity and specificity of these new proteins can also be improved by linking more fingers together or by designing proteins that bind as dimers and thus recognize an extended site. These new proteins can then be modified by adding other domains--for activation or repression of transcription, for DNA cleavage, or for other activities. Such designer transcription factors and other new proteins will have important applications in biomedical research and in gene therapy.
Assuntos
Proteínas/química , Proteínas/metabolismo , Dedos de Zinco , Motivos de Aminoácidos , Animais , Sítios de Ligação , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Especificidade por SubstratoRESUMO
Protein-protein interactions often play a crucial role in stabilizing protein-DNA complexes and thus facilitate site-specific DNA recognition. We have worked to incorporate such protein-protein contacts into our design and selection strategies for short peptide extensions that promote cooperative binding of zinc finger proteins to DNA. We have determined the crystal structure of one of these fusion protein-DNA complexes. The selected peptide extension was found to mediate dimerization by reaching across the dyad axis and contacting a hydrophobic patch on the surface of the zinc finger bound to the adjacent DNA site. The peptide-zinc finger protein interactions observed in this structure are similar to those of some homeodomain heterodimers. We also find that the region of the zinc finger surface contacted by the selected peptide extension corresponds to surfaces that also make key interactions in the zinc finger proteins GLI and SWI5.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Dedos de Zinco , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalografia por Raios X , DNA/genética , Proteínas de Ligação a DNA/genética , Dimerização , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Peptídeos/genética , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
We have determined the crystal structure of a complex containing the engrailed homeodomain Gln50 --> Ala variant (QA50) bound to the wild-type optimal DNA site (TAATTA) at 2.0 A resolution. Biochemical and genetic studies by other groups have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains (distinguishing among various sites of the general form TAATNN). However, biochemical studies of the QA50 variant had revealed that it binds almost as tightly as the wild-type protein and with only modest changes in specificity. We have now determined the crystal structure of the QA50 variant to help understand the role of residue 50 in site-specific recognition. Our cocrystal structure shows some interesting changes in the water structure at the site of the substitution and shows some changes in the conformations of neighboring side chains. However, the structure, like the QA50 biochemical data, suggests that Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain.
Assuntos
DNA/química , Proteínas de Homeodomínio/química , Cristalografia , DNA/metabolismo , Glutamina/química , Proteínas de Homeodomínio/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/química , Conformação Proteica , TATA BoxRESUMO
Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Proteoma , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Masculino , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Ratos , Ratos Sprague-DawleyRESUMO
We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.
Assuntos
Poluentes Atmosféricos/toxicidade , Aeronaves , Nefropatias/induzido quimicamente , Rim/efeitos dos fármacos , Petróleo/toxicidade , Proteínas/metabolismo , Aerossóis , Aminopeptidases/metabolismo , Animais , Proteínas do Citoesqueleto , Eletroforese em Gel Bidimensional , Proteínas de Grupo de Alta Mobilidade/metabolismo , Querosene/toxicidade , Rim/metabolismo , Masculino , Camundongos , Exposição Ocupacional , Fator 2 de Elongação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tropomiosina/metabolismoRESUMO
We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.
Assuntos
Hidrocarbonetos/toxicidade , Pulmão/efeitos dos fármacos , Exposição Ocupacional , Proteoma , Animais , Eletroforese em Gel Bidimensional , Pulmão/metabolismo , Pulmão/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Lead is a potent neuro- and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead-induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of lead's renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two-dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either or) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix-assisted laser desorption/ionization mass spectrometry/electrospray ionization-mass spectrometry (MALDI-MS/ESI-MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of lead's nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.
Assuntos
Rim/metabolismo , Chumbo/farmacologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Rim/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.
Assuntos
Citosol/química , Córtex Renal/ultraestrutura , Medula Renal/química , Proteínas/análise , alfa-Globulinas/análise , Animais , Argininossuccinato Sintase/análise , Calbindinas , Calcineurina/análise , Eletroforese em Gel Bidimensional , Glutationa Transferase/análise , Masculino , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transcetolase/análiseRESUMO
The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA cis-element that is required for high-level expression of viral surface gene transcripts and appears to function by activating mRNA export to the cytoplasm. We have previously shown that multiple fragments of the PRE bind to two cellular proteins of approximately 35 and 55 kDa in molecular mass and that this binding correlates with function. By a combination of column chromatographic techniques and SDS-polyacrylamide gel electrophoresis, we have been able to purify the smaller protein. Amino-terminal sequencing of the purified protein shows identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an RNA-binding glycolytic enzyme that has been implicated in the export of tRNA. Immunoprecipitation analysis reveals that GAPDH is indeed present in the protein-RNA complex resulting from incubation of crude nuclear extracts with a functional region of the PRE. Furthermore, binding of the cellular 35 kDa protein to the PRE fragment is blocked by NAPDH, as would be expected for RNA binding by GAPDH. Finally, purified commercial GAPDH also binds specifically to this RNA fragment. Therefore, GAPDH is one of the cellular proteins that binds to the PRE, and may be involved in the posttranscriptional regulation of hepatitis B virus gene expression.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Vírus da Hepatite B/fisiologia , RNA Viral/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Vírus da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Transcrição GênicaRESUMO
The crystal structure of Dps, a DNA-binding protein from starved E. coli that protects DNA from oxidative damage, has been solved at 1.6 A resolution. The Dps monomer has essentially the same fold as ferritin, which forms a 24-mer with 432 symmetry, a hollow core and pores at the three-fold axes. Dps forms a dodecamer with 23 (tetrahedral) point group symmetry which also has a hollow core and pores at the three-folds. The structure suggests a novel DNA-binding motif and a mechanism for DNA protection based on the sequestration of Fe ions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Ferritinas/química , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/fisiologia , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Estresse Oxidativo , Mutação Puntual , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Crystal structures of the Mahoney strain of type 1 poliovirus complexed with the antiviral compounds R80633 and R77975 were determined at 2.9 A resolution. These compounds block infection by preventing conformational changes required for viral uncoating. In various drug-poliovirus complexes reported earlier, no significant conformational changes were found in the structures of the capsid proteins. In the structures reported here, the strain of virus is relatively insensitive to these antivirals. Correspondingly, significant conformational changes are necessary to accommodate the drug. These conformational changes affect both the immediate vicinity of the drug binding site, and more distant loops located near the fivefold axis. In addition, small but concerted shifts of the centers of mass of the major capsid proteins consistently have been detected whose magnitudes are correlated inversely with the effectiveness of the drugs. Collectively, the drug complexes appear to sample the conformational repertoire of poliovirus near equilibrium, and thus provide a possible model for the earliest stages of viral uncoating during infection.
RESUMO
DA strain of TMEV induces a chronic, persistent, demyelinating disease in SJL/J weanling mice, while inoculation with GDVII strain of TMEV induces an acute, lethal neurovirulent disease. We show that three amino acids in the DA EF loop-DAV P2 141 Lys, 143 Gly, and 173 Thr-are part of a neutralization site of DA monoclonal antibody (mAb), DAmAb1. DA virus with a mutation of VP2 143 from Gly to Asp, like wild-type virus, persists 6 weeks postinfection (PI) and produces white matter disease. DA virus with a mutation of VP2 141 from Lys to Asn persists but does not induce significant white matter disease. DA virus with a mutation of DA VP2 173 from Thr to Phe fails to persist or to induce significant white matter disease. The diversity and complexity of the mutant virus-induced disease phenotype presumably reflects the varied effects of the mutated amino acid residues on the three-dimensional structure of the viral capsid. The localization of DA VP2 141 and VP2 173 near the putative receptor binding region of the virus suggest that a disruption of interactions between the virus and its receptor is important in the late demyelinating disease and for virus neutralization.
Assuntos
Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Epitopos de Linfócito B/imunologia , Poliomielite/imunologia , Theilovirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Capsídeo/química , Capsídeo/genética , Proteínas do Capsídeo , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Epitopos de Linfócito B/análise , Camundongos , Mutagênese Sítio-Dirigida , Testes de Neutralização , Poliomielite/patologia , Poliomielite/virologia , Conformação Proteica , Medula Espinal/patologia , Theilovirus/genética , Theilovirus/patogenicidadeRESUMO
We describe the effect of deamination of lysine and blocking of arginine residues on the assembly of collagen into native fibrils and SLS aggregates. Treatment of collagen solutions with one or both of these procedures does not prevent the formation of fibrils or SLS aggregates but reduces their ability to form assemblies with accurate longitudinal registration. These observations provide direct confirmation that hydrophobic interactions are important in collagen assembly. Unbanded fibrils were formed within the first 24 h at 4 degrees C from both acidic and neutralized deaminated and from neutralized control collagen solutions, transversely banded fibrils appearing later. This is compatible with the suggestion that initially, collagen fibrils are assembled by lyotropic liquid crystallization and with other observations which suggest that collagen molecules are initially free to move laterally within the fibril before being locked into place. Fibrils assembled from deaminated collagen solution show two variant longitudinal registration patterns which grade into one another. This suggests that, with a reduction in positively charged side chains, the thermodynamic energy minima responsible for longitudinal registration are less sharp compared with control collagen solutions. Reduction of positive charge by chemical modification helps to explain why the chemical modifications reduce swelling of collagen fibres. It also helps to explain why fibrils form spontaneously at 4 degrees C in both arginine-blocked and deaminated collagen solutions. Thus chemical modifications of rat tail tendon provides new insight into the mechanisms in collagen assembly.
Assuntos
Arginina/química , Colágeno/química , Lisina/química , Tendões/química , Acetatos , Ácido Acético , Animais , Desaminação , Eletroquímica , Ratos , Solubilidade , Cauda , Extratos de Tecidos , Água/químicaRESUMO
An improved method for expression of poly-beta-hydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus has been developed using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) in BTI-TN-5B1-4 Trichoplusia ni cells which results in high level production of active PHA synthase. Confirmation of expression of authentic PHA synthase was obtained by Western analysis which also revealed the presence of several apparent proteolytic cleavage products. N-terminal sequence data were obtained from the 64-kDa protein which verified its identity. The PHA synthase produced in this system constitutes approximately 50% of total protein after 60 h of viral infection and is found approximately equally distributed in both soluble and membrane-associated fractions. The expression level allowed rapid purification of the soluble form of PHA synthase to approximately 90% homogeneity in a single liquid chromatography step on hydroxylapatite. Using a direct spectrophotometric assay, analyses show that the enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation between enzyme concentration and specific activity. Over 1000 units of soluble enzyme were obtained from a 250-ml culture of T. ni cells with an apparent initial specific activity of 12 mumol min-1 mg-1. The amount of PHA synthase activity is significantly higher than previously obtained from much larger bacterial cultures. The method described here should provide a general approach for the expression of active PHA synthases from a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing proteins.