AMP deaminase from rat brain: Purification and characterization of multiple forms

Abstract

1. 1.|When brain extracts were chromatographed on a phosphocellulose column, using a NaCl gradient for elution, three peaks and a shoulder with AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) activity were obtained. They were designated as Peaks I, II, III and IV in the order of elution from the column. When Peaks I, II and IV were individually applied to the phosphocellulose column, each was chromatographed as a single peak at the same position as that for the original peak and the mixtures of them yielded only the peaks corresponding to the parental peaks. 2. 2.|Deaminase Peaks I, II and IV were purified by column chromatography using phosphocellulose and DEAE-cellulose. About 130-fold purification was obtained for Peak I, 280-fold for Peak II and 770-fold for Peak IV. 3. 3.|A broad optimum pH was observed in Peak I and a sharp optimum pH, at pH 6, in Peak IV. Peak I was rapidly inactivated when heated at 50 °C. Under the same condition, Peaks II and IV were inactivated more slowly. The rates of deamination of dAMP, relative to AMP, in three deaminase preparations are different from one another. 4. 4.|The molecular sizes of three deaminases are similar. 5. 5.|Deaminase Peak I has apparently a higher affinity for AMP than other types of deaminase both in the absence and presence of activators, while Peak IV has the lowest affinity of all. 6. 6.|The regulatory properties of these deaminases are similar. The velocity vs AMP concentration curves are sigmoidal in the absence of activator such as ATP, KCl or NaCl and the curves become nearly hyperbolic in the presence of activator. The maximum velocities were the same both in the absence and presence of activator, while the enzymes showed a higher affinity for AMP in the presence of activator.