STUDIES ON HISTIDINE DEAMINASE***

Abstract

From liver of the rabbit to which L-histidine.HCl(0.5 g/kg) had been previously administered, histidine deaminase was purified as follows: grind, mix with 5 vol. of water at 0-5[degree]C for 1 hr., centrifuge at 300 rpm for 20 min., adjust the supernatant fluid to pH 5.2 with 4% acetic acid, centrifuge at 3000 rpm for 30 min., dissolve the precipitate (ppt.) in a minimal amount of water, adjust to pH 7.2 with 4% NaHCO3, fractionate with ammonium sulfate between 0.3 and 0.5 saturation, dissolve ppt. in water, adsorb on Ca phosphate gel at 0-5[degree]C for 30 min., elute with 01M phosphate buffer(pH 8.3), and centrifuge at 3000 rpm for 10 min. This supernatant fluid regains its enzymic activity by the simultaneous addition of 3.8x10-4 [image] folic acid and 3.3x10-3 [image] reduced glutathione-(GSH). The preparation at the stage of ammonium sulfate fractionation is completely inhibited by 10-4 [image] EDTA(ethylene-diamine tetraacetate), and the EDTA inactivation is counteracted strongly by 3.3x10-4M Co++, Zn++, Mn++, Cd++ in that order. When this crude preparation is submitted to purification as in the above procedure, a completely inactive preparation is obtained, which can be reactivated by the simultaneous addition of 7.6x10-4 [image] folic acid, 1.1x10-3 [image] GSH, and 3.3x10-4 [image] Co++. Co++ cannot be replaced by Ca++, which is indispensable in case of the guinea pig liver enzyme. The enzymatic decomposition of L-histidine is urocanic acid.