Purification and reversible inactivation of the isocitrate dehydrogenase from an obligate halophile.
The nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase of Halobacterium cutirubrum is rapidly inactivated at low NaCl levels. As much as 75% of the initial activity can be restored by dialyzing the inactive enzyme against 4 m NaCl. A mixture of 4 mm isocitrate and 10 mm MnCl(2) gives the same protection as 4 m NaCl but does not replace the NaCl requirement for reactivation. The reactivated and native enzymes have identical sedimentation rates on sucrose gradients, electrophoretic mobilities on polyacrylamide gels, and elution rates from Sephadex G-200. However, there are distinct differences between the active and inactive forms of the enzyme. Compared with the active enzyme, the inactive protein has a lower sedimentation rate, a lower electrophoretic mobility, and a faster elution rate from Sephadex. These differences indicate that inactivation causes a major conformational change in the protein. Presumably, the removal of NaCl permits the enzyme to expand into a less dense, inactive form. The isocitrate dehydrogenase was purified 69-fold by a procedure involving the following steps. When the enzyme is selectively protected with isocitrate and MnCl(2) at low ionic strength, most of the contaminating proteins are precipitated with (NH(4))(2)SO(4) at 0.9 saturation. The enzyme in the supernatant fluid is then inactivated at low NaCl levels, precipitated with 0.5 saturated (NH(4))(2)SO(4), and reactivated with 4 m NaCl. Minor impurities are removed by gel filtration on Sephadex G-200. The resulting preparation is more than 95% pure as judged by disc electrophoresis.
D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002712:Chlorides; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D006217:Halobacterium / Q000201:enzymology*; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification; D008274:Magnesium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology*
D002712:Chlorides; D012965:Sodium Chloride; D007521:Isocitrate Dehydrogenase; D008274:Magnesium