RESUMO
We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rdlrd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAP1 and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAP1 levels are reduced by more than 90%. The specific downregulation of GCAP1 in rdlrd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica/fisiologia , Retina/química , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/análise , Galinhas , Clonagem Molecular , DNA Complementar/genética , Proteínas Ativadoras de Guanilato Ciclase , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1).
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cromossomos Humanos Par 6 , Guanilato Ciclase/metabolismo , Células Fotorreceptoras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , Ativação Enzimática , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Doenças Retinianas/genética , Homologia de Sequência de AminoácidosRESUMO
Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.
Assuntos
Aminoácido Oxirredutases/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Olho , Guanilato Ciclase/metabolismo , Lipoproteínas , Receptores de Detecção de Cálcio , Segmento Externo da Célula Bastonete/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Calmodulina/química , Bovinos , Clonagem Molecular , Sondas de DNA/química , Ativação Enzimática , Hipocalcina , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neurocalcina , Óxido Nítrico Sintase , Fragmentos de Peptídeos/química , Células Fotorreceptoras/metabolismo , Filogenia , Ranidae , Recoverina , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de FluorescênciaRESUMO
A spectrofluorimetric procedure for the determination of indole-3-acetic acid, indole-3-propanoic acid and indole-3-butyric acid by derivatization with copper sulphate-sulphuric acid solution has been developed. The optimum reaction conditions, the effect of interferents and the advantages associated with the use of first- and second-derivative synchronous spectrofluorimetry have been studied. The detection limits are 3, 12 and 6 ng/ml for indole-3-acetic, indole-3-propanoic and indole-3-butyric acid, respectively.
