Comparison of the primary structure of nuclear and mitochondrial glutamate dehydrogenase from bovine liver.
Glutamate dehydrogenase (GDH) from bovine liver nuclei was compared to bovine liver mitochondrial GDH. The nuclei were isolated in sucrose buffer and sonicated, and glutamate dehydrogenase activity was extracted with 0.1 M potassium phosphate buffer. The enzyme was purified by ammonium sulfate fractionation, heating, gel filtration, affinity chromatography, and absorption chromatography to homogeniety. Nuclear GDH had the same apparent molecular weight on SDS-PAGE as mitochondrial GDH. The overall charge was slightly more negative. Cyanogen bromide and tryptic peptides of bovine nuclear and mitochondrial glutamate dehydrogenase were separated by HPLC reverse-phase chromatography using a linear gradient of 0-60% acetonitrile. Only about half of the nuclear and mitochondrial peptides had the same retention time. Several nuclear peptides from the tryptic digest were sequenced. Eight of the amino acids differed from the published sequence of mitochondrial GDH (of 99 that were sequenced). The amino acid composition of one peptide was determined and it contained 4 (of 37 amino acids) that were different from the published composition of the corresponding peptide from bovine mitochondrial GDH. The composition data agree with the sequence data from this peptide. We conclude that GDH does exist in bovine liver nuclei and that it probably differs by less than 10% in amino acid sequence from mitochondrial GDH.
McDaniel
H G
HG
VAMC, Birmingham, Alabama, USA.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase