RESUMO
Four independent phage clones containing the fragments of cone-specific cGMP phosphodiesterase (PDE) alpha' subunit (PDE-alpha') cDNA were isolated from the human cDNA library. The screening of the genomic library resulted in isolation of four independent phage clones with the fragments of human cone PDEalpha' gene including 5'-flanking region and exons ranged from 1 to 14 (overall 32 kilobases). Structural studies of the clones made it possible to establish the complete human cone PDEalpha' cDNA structure (3455 base pairs). The encoding polypeptide consists of 858 amino acid residues with a calculated molecular mass of 99169 Da. The deduced amino acid sequence displays high homology to the earlier analyzed catalytic alpha, beta and alpha' subunits of bovine, human, chicken and mouse photoreceptor PDEs.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Células Fotorreceptoras Retinianas Cones/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Galinhas , Clonagem Molecular , DNA Complementar/química , Éxons , Biblioteca Gênica , Biblioteca Genômica , Humanos , Íntrons , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Células Fotorreceptoras/enzimologia , Mapeamento por Restrição , Retina/enzimologia , Homologia de Sequência de AminoácidosRESUMO
The recombinant and 21 mutant phosphodiesterase (PDE) gamma subunit (PDE gamma) genes were expressed by sequential transcription and translation in vitro. Inhibitory properties of these mutants and their interactions with PDE catalytic and transducin alpha subunits were studied. The interaction of the PDE gamma subunit with the catalytic ones proceeds in two steps--primary binding and inhibition. The central region of the PDE gamma molecule enriched with the basic amino acid residues (particularly, Lys-29, Lys-31 and Arg-33), is involved in the primary binding, and the PDE gamma C-terminus plays the key role in inhibition. The spatial orientation of the C-terminus is of great importance here. The PDE gamma C-terminus also affects binding to catalytic moieties.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Segmento Externo da Célula Bastonete/enzimologia , Transducina/química , Aminoácidos/química , Animais , Sequência de Bases , Catálise , Bovinos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Conformação ProteicaRESUMO
The alpha-subunit primary structure of cyclic GMP phosphodiesterase has been determined by parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The enzyme alpha-subunit contains 858 amino acid residues, its N-terminal amino group being acetylated. The partial primary structure of the enzyme beta-subunit has also been elucidated. A significant homology has been found between the alpha- and beta-subunits of cGMP phosphodiesterase.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Retina/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA/genética , Dados de Sequência Molecular , Iniciação Traducional da Cadeia PeptídicaRESUMO
The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated.
