The isoenzymes of isocitrate dehydrogenase in Acinetobacter lwoffi.

Preliminary results had indicated that the isocitric dehydrogenase (IDH) of the bacterium Acinetobacter lwoffi 4B was activated by low levels of glyoxylate. Furthermore, in the presence of glyoxylate the enzyme had been found to be more stable to heat or high concentrations of urea. Subsequent studies have shown that two isoenzymes of NADP-linked IDH exist in A. lwoffi and that only one of these is stimulated and protected by glyoxylate. It has also been shown that pyruvate has a similar activating and protecting effect as that of glyoxylate. On addition of either of these effectors the activity of the sensitive enzyme is markedly changed in its dependence on substrate concentration and pH (the former resulting from changes in both the apparent Km and Vm for the substrates). The isoenzymes have been separated by DEAE-cellulose chromatography, cellulose acetate electrophoresis, gel-filtration and preparative rate zonal ultracentrifugation. Investigations using the latter two techniques have shown the sensitive enzyme to be a much larger molecule than the insensitive one. The use of preparative zonal ultracentrifugation for the separation of isoenzymes was novel and these studies have shown it to be a potentially powerful technique for both the separation and purification of isoenzymes. Studies on the separated isoenzymes have shown them to differ in their sensitivities to heat and urea and to have different dependences on pH, temperature and substrate concentration. Experiments undertaken with the aim of elucidating the physiological role of both isoenzymes are also described and discussed.