Factors Influencing the Activity and the Stability of IMP Dehydrogenase of Rat Liver

Abstract

IMP dehydrogenase is the key enzyme in the biosynthesis of guanine nucleotides, and is known to have close relationship with many biological phenomena including malignant transformation. Its inhibitors have been utilized in attempt to interrupt the growth of cancer cells and viruses. This experiment was performed to define the conditions for assay of IMPO activity and puriftcation of the enzyme by elucidating the effect of various factors on its activity and stability. The enzyme was prepared from partially hepatectomized rat liver by treatment with ammonium sulfate and OEAE-celluiose chromatography. The enzyme exhibited maxium activity at pH 7.0 and sharp decrease in its activity below and above pH 7.0. The enzyme required free sulfhydryl groups such as mercaptoethanol and dithioerythritol for its full activity, and mercaptoethanol inhibited 1MPO activity at the concentration above 10 mM. Among the monovalent cations tested, both K+ and Na + manifested activating effect in contrast with the inhibitory effect of Li+, and the activating effect of sodium was only about half the effect of potassium. All the divalent cations such as Mg2 '. Ca2 +, Cu2 +, Zn2 + exhibited strong inhibitory effect on IMPO activity. EOTA was added to the buffers to inhibit various phosphatases and also to remove any trace divalent actions, and did not exert any inhibitory effect on IMPO activity. Glycerol added to purification buffers to mimic intracellular environment manifested slightly inhibitory effect at 10% concentration, but the ethanol showed relatively strong inhibition. As the duration of dialysis increased, the IMPD activity was increased proportionally until 24 hours. And ammonium sulfate used for salting out IMPD also showed strong inhibitory effect, therefore complete removal of the salt are required before assaying the activity. When IMPD preparation was stored at ~60'C. ~20C. 4°C, and 25°C for 7 days, only the preparation stored at ~ 60"C retained most of the initial activity, and that stored at 25°C lost most of its activity in 24 hours. Thiols such as dithioerythritol and mercaptoethanol exhibited stabilizing effect on IMPD stored at 4('C, and K+, glycerol, and EDTA did not show any protective effect Change of Tris buffer (pH 7.4) buffer neither to phosphate nor to pH 8.0 improved the stability of IMPD.