title: Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis.
abstract: Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, 65211, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Karr
authors.forename: D W
authors.initials: DW
authors.lastname: Emerich
mesh_terms: D000595:Amino Acid Sequence; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid
chemical_list: D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D008274:Magnesium; D002118:Calcium