title: Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene.
abstract: 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.
authors.affiliation: Laboratoire de Biologie et Physiologie de la Reproduction et du Développement, Faculté des Sciences I, BP5366, Maarif, Casablanca, Morocco.
authors.forename: Khadija
authors.initials: K
authors.lastname: Mounaji
authors.forename: Nour-Eddine
authors.initials: NE
authors.lastname: Erraiss
authors.forename: Abdelghani
authors.initials: A
authors.lastname: Iddar
authors.forename: Maurice
authors.initials: M
authors.lastname: Wegnez
authors.forename: Aurelio
authors.initials: A
authors.lastname: Serrano
authors.forename: Abdelaziz
authors.initials: A
authors.lastname: Soukri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D017125:Genes, Protozoan; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D010999:Pleurodeles; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases