Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast,Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Peroxisomal NADP-linked isocitrate dehydrogenase (Ps-NADP-IDH) was purified for the first time from Candida tropicalis cells grown on n-alkane as a carbon source, which was effective in proliferation of peroxisomes. The properties of Ps-NADP-IDH were compared with those of mitochondrial NAD-linked isocitrate dehydrogenase (Mt-NAD-IDH) purified from the cells grown on acetate, in which peroxisomes did not proliferate. Ps-NADP-IDH was a homodimer of identical subunits (45 kDa), while Mt-NAD-IDH was suggested to be a heterooctamer composed of two types of subunits with different molecular masses (41 and 38 kDa). Kinetic studies revealed that Ps-NADP-IDH gave Michaelis-Menten saturation curves against isocitrate and NADP concentrations, whereas Mt-NAD-IDH was an allosteric enzyme regulated by ATP, AMP, and citrate. Inhibition by 2-oxoglutarate, a precursor of glutamate, was observed only for Ps-NADP-IDH. Both enzymes were inhibited by concomitant addition of oxalacetate and glyoxylate. The function of Ps-NADP-IDH seems to be completely discriminated from that of Mt-NAD-IDH as reflected by their distinct subcellular localizations. Furthermore, the properties of Ps-NADP-IDH were also compared with those of other mitochondrial and cytosolic IDHs from sources reported previously.     

Characterization of the catalase of the n-alkane-utilizing yeast Candida tropicalis functionally expressed in Saccharomyces cerevisiae

Candida tropicalis catalase (CTC) genomic DNA was recombined on a plasmid with the galactose-inducible GAL7 promoter and expressed highly as a heme protein in Saccharomyces cerevisiae as host. The percentage of recombinant CTC (rCTC) in total extractable protein amounted to at least 25%. The rCTC was purified and characterized in terms of subunit mass, behavior in native polyacrylamide gel electrophoresis, absorption spectrum, amino-terminal amino acid sequence, peptide map, specific activity, and Michaelis constant (K _m) value for hydrogen peroxide. These properties were similar or identical to those of the purified enzyme from C. tropicalis (CTC). From these results, this system appears suitable for high expression of functional catalase protein having heme. Correspondence to: A. Tanaka     

Activity and Stability of a New Sweet Protein with Taste-modifying Action, Curculin

Curculin elicited a sweet taste. After the sweetness of curculindiminished, application of deionized water or an acid to thetongue induced a sweet taste. The maximum sweetness of curculinitself was equivalent to thesweetness of 0.35 M sucrose. Themaximum sweetness induced by 0.02 M citric acid or deionizedwater after curculin dissolved in a buffer of pH 6.0 was heldin mouth for 3 min was also equivalent to that of 0.35 M sucrose.The sweetness induced by deionized water was completely suppressedby the presence of 1 mM CaCl₂ or MgCl₂, while that induced byan acid was not suppressed by the presence of divalent cations.Based on these results, the mechanism of the taste-modifyingactivity was discussed. Stability of curculin was examined undervarious conditions. The taste-modifying activity of curculinwas unchanged when curculin was incubated at 50°C for 1h between pH 3 and 11.