RESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Metais/farmacologia , Pleurodeles/metabolismo , Xenopus laevis/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Sítios de Ligação , Cádmio/metabolismo , Cádmio/farmacologia , Células Cultivadas , Cobre/metabolismo , Cobre/farmacologia , DNA Complementar/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Modelos Moleculares , Dados de Sequência Molecular , Pleurodeles/genética , Estrutura Terciária de Proteína , Alinhamento de Sequência , Xenopus laevis/genética , Zinco/metabolismo , Zinco/farmacologiaRESUMO
The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Músculo Esquelético/enzimologia , Pleurodeles , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia em Gel , DNA/análise , Eletroforese em Gel de Poliacrilamida , Genes de Protozoários , Gliceraldeído-3-Fosfato Desidrogenases/genética , Focalização Isoelétrica , Dados de Sequência Molecular , Peso Molecular , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The characterization of metallothionein in the Urodele amphibian species Pleurodeles waltl was achieved. A simple and rapid method for identification of metallothionein, based on its strong affinity for cadmium (109Cd), was used. We were able to show that metallothionein is constitutively synthesized in liver, ovary and brain. The property of metallothionein to strongly bind essential (Zn, Cu) as well as toxic (Cd, Hg) metals is consistent with a dual role in cellular metabolism, ie. homeostatis and detoxification of heavy metal ions.