Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis.
2-Oxoglutarate decarboxylase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. It had a molecular weight of 250,000 and consisted of four identical subunits of molecular weight 62,000. The enzyme was specific for 2-oxoglutarate, but not for other 2-oxo acids such as pyruvate and oxalacetate. Thiamin pyrophosphate and MgCl2 were required for maximum activity. The Km values of the enzyme for 2-oxoglutarate, thiamin pyrophosphate, and MgCl2 were 330, 56, and 93 microM, respectively. 2-Mercaptoethanol and NADP+ augmented significantly the enzyme activity. The amino acid composition and amino acid sequence of the amino-terminal region of 2-oxoglutarate decarboxylase were determined. On ouchterlony double-immunodiffusion gels, the anti-2-oxoglutarate decarboxylase antibody gave sharp precipitin lines against the mitochondrial fraction of E. gracilis and the purified 2-oxoglutarate decarboxylase, but not against pyruvate decarboxylase from Saccharomyces cerevisiae. On Immunoblots of the crude extract of Euglena, the antibody recognized two polypeptides whose molecular weights were 62,000 and 65,000, respectively. The polypeptide with the molecular weight of 62,000 was found only in mitochondrial fractions. In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in Euglena cells.
Shigeoka
S
S
Department of Food and Nutrition, Kinki University, Nara, Japan.
Nakano
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005056:Euglena gracilis / Q000201:enzymology*; D007120:Immunochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011487:Protein Conformation
D000596:Amino Acids; D002262:Carboxy-Lyases; C021474:2-oxoglutarate decarboxylase