RESUMO
An ATX1 homologue of 503 bp length was cloned from a rat cDNA library, and the deduced protein from the cDNA was found to contain 68 amino acids with a predicted molecular mass of 7.2 kDa. The rat ATX1 homologue protein (Rah1p), which shows 35%, 38%, and 89% identities with Atx1p, CUC-1, and HAH1, respectively, conserves both the MTCXXC copper-binding site in the N terminus and the KTGK lysine-rich region in the C terminus. In Northern blot analysis, rah1 mRNA was found to be expressed at high levels in the liver, small intestine, and testis. Expression of rah1 cDNA complemented a null atx1 mutant strain in yeast. Thus, Rah1p was concluded to be a functional copper chaperone.
Assuntos
Proteínas de Transporte , Proteínas Fúngicas/genética , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cobre/metabolismo , Proteínas de Transporte de Cobre , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Teste de Complementação Genética , Masculino , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.
Assuntos
Chaperonas Moleculares/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Clonagem Molecular , DNA Complementar , Glutationa Transferase/metabolismo , Humanos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. We show here that, in addition to ATPase activity, purified Hsp70 free from nucleoside-diphosphate (NDP) kinase exhibits intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis were in a similar range at the optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP. Hsp70 exhibited a considerably strict preference for ATP as a phosphate donor, and a biased substrate specificity, unlike NDP kinase; ADP, UDP, CDP > dTDP, dCDP > GDP, dGDP. During the reaction, Hsp70 formed an acid-labile autophosphorylated intermediate, and nucleoside diphosphate-dependent dephosphorylation of the latter then occurred. These properties of Hsp70 are not identical but similar to those of NDP kinase, but are not similar to those of adenylate kinase and ATP synthase.