NADP(+)-activable, NAD(+)-specific glutamate dehydrogenase. Purification and immunological analysis.
An NAD(+)-specific glutamate dehydrogenase (NAD-GDH) that is inducible by L-glutamine was isolated from Achlya klebsiana and purified to electrophoretic homogeneity. The enzyme is only partially active in vitro unless NADP+ (an activator) is present in both its oxidative deamination and reductive amination reactions. This type of enzyme was reported (LéJohn, H.B. (1971) Nature 231, 164-168) to be widespread among the amorphous group of algae-related fungi classified as Oomycota. The enzyme retained its dependence on NADP+ at all stages of its purification. NADP+ decreased the Km of substrates 3-fold and increased the Vmax 4-fold. M(r) of the undernatured enzyme was 480,000, and, denatured, only a single subunit of M(r) 120,000 was seen. A polyclonal antibody raised in rabbit against purified enzyme subunit excised from SDS-polyacrylamide gel electrophoresis gels immunoprecipitated the M(r) 120,000 polypeptide, the undenatured enzyme, and a physically distinct polypeptide of M(r) 74,000. The antibody, purified against the M(r) 120,000 enzyme subunit as anchored antigen on Sepharose, still immunoprecipitated the M(r) 74,000 polypeptide. The M(r) 74,000 polypeptide was found to be a subunit of a M(r) 220,000 native protein.
Yang
B
B
Manitoba Institute of Cell Biology, Winnipeg, Canada.
LéJohn
H B
HB
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification*; D005800:Genes, Fungal; D005969:Glutamate Dehydrogenase / Q000276:immunology / Q000302:isolation & purification*; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009868:Oomycetes / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase