RESUMO
Aging is generally defined and studied as a population phenomenon. However, there is great interest, especially when discussing human aging, in the identification of factors that influence the life span of an individual organism. The nematode Caenorhabditis elegans provides an excellent model system for the study of aging at the level of the individual, since young nematodes are essentially clonal yet experience a large range of individual life spans. We are conducting gene expression profiling of individual nematodes, with the aim of discovering genes that vary stochastically in expression between individuals of the same age. Such genes are candidates to modulate the ultimate life span achieved by each individual. We here present statistical analysis of gene expression profiles of individual nematodes from two different microarray platforms, examining the issue of technical vs. biological variance as it pertains to uncovering genes of interest in this paradigm of individual aging.
Assuntos
Envelhecimento/genética , Caenorhabditis elegans/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Biomarcadores/análise , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Genes de Helmintos/genética , Variação Genética/genética , Proteínas de Fluorescência Verde/análise , Humanos , Substâncias Luminescentes/análise , Modelos Animais , Processos EstocásticosRESUMO
Advances in understanding of mitochondrial physiology and genetics in relation to pathology have exploded in the last decade. Paralleling this increase has been an active debate about the role of mitochondrial oxidative stress with regard to mitochondrial DNA mutations, aging, and disease. We discuss in a historical context the rapid progress in our understanding of the role of mitochondrial DNA mutations in disease, mitochondrial oxidative stress in aging, and the potential interplay between these two phenomena.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/fisiologia , Mutação , Estresse Oxidativo/fisiologia , Animais , Predisposição Genética para Doença/genética , HumanosRESUMO
We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Streptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an "on-slide" sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or yn ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.
Assuntos
Sulfato de Amônio/química , Peptídeos/análise , Pronase/química , Hormônio Adrenocorticotrópico/química , Sequência de Aminoácidos , Bradicinina/química , Hidrólise , Indicadores e Reagentes , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptídeos/química , Salmonella/química , Análise de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.
Assuntos
Adenosina Trifosfatases/metabolismo , Arginina/metabolismo , Proteínas de Transporte , Proteínas de Membrana/metabolismo , Mitocôndrias Hepáticas/enzimologia , Oligomicinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Corpos de Inclusão/enzimologia , Corpos de Inclusão/metabolismo , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , ATPases Mitocondriais Próton-Translocadoras , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Desnaturação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de FluorescênciaRESUMO
Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside.