Isozymes of Isocitrate Dehydrogenase from an Obligately Psychrophilic Bacterium, Vibrio Sp. Strain ABE-1: Purification, and Modulation of Activities by Growth Conditions

Abstract

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.

Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria

“…Then, these His-tagged wild-type and mutated IDH proteins were overexpressed in the E. coli DEK2004 cells and were purified as described above. From SDS-PAGE of the final elutant of Ni-NTA column chromatography, the purified enzyme samples were found to contain a major protein with a molecular mass of about 80 kDa (data not shown), corresponding to those of the CmIDH and CpIDH proteins (Ishii et al 1987;Maki et al 2006). In this study, a protease inhibitor, PMSF, was added at several times during the purification.…”

“…On the basis of subunit structure, bacterial NADP + -specific IDHs can be classified into two types, homodimer consisting of about 40-45 kDa subunits and monomer with a molecular mass of 80-100 kDa. Many bacteria possess only one of either type IDH, but both of the two type IDHs are known to be present in several bacteria such as Colwellia maris (Ochiai et al 1979;Ishii et al 1987), Colwellia psychrerythraea NRC1004 (Maki et al 2006), Ralstonia eutropha (Wang et al 2003) and a psychrotrophic bacterium, Pseudomonas psychrophila (Matsuo et al 2010). …”

Isolation and characterization of a mutant defective in one of two isozymes of the isocitrate dehydrogenase from a psychrophilic bacterium Vibrio sp. strain ABE-1.

“…Crude extract was prepared from sonically disrupted cells by a method similar to one described previously (11) with buffer consisting of 20 mM Tris-HC1 (pH 8.4 at 20°C), 0.5M NaCI, 1 mM MnCl2, 1 mM EDTA and 10 mM 2-mercaptoethanol. Enzyme activities for IDH-I and -II were measured spectrophotometrically as previously described (I1).…”

“…IDH-I and IDH-II were purified from strain ABE-1 as described previously (11). Anti-IDH-I and IDH-II antibodies were prepared from the serum of a rabbit immunized against purified IDH-I and IDH-II, respectively.…”

“…OcHIA1 et al (7,10) reported that both types of IDH were coexistent in this bacterium and the dimeric enzyme (IDH-I) was thermostable while the monomeric one (IDH-II) was extremely thermolabile. Recently, we homogeneously purified both IDHs and characterized them in more details (11). IDH-I and -II have similar amino acid composition but are different in immunocross-reactivity.…”