Purification and characterization of an endogenous inhibitor of alcohol dehydrogenase from maize root

Abstract

An inhibitor of alcohol dehydrogenase (ADH) in the maize root has been purified by a stepwise method consisting of gel filtration, ion-exchange and hydroxylapatite chromatography. The molecular weight of the inhibitor is approx. 10 000 as estimated by gel filtration and ultrafiltration techniques. The inhibitor is sensitive to heat although boiling for 5 min does not completely destroy its activity. The inactivation of ADH by the inhibitor is dependent on the level of substrate ADH and the level of the inhibitor. The reaction is also time dependent. The inhibitor shows its optimum activity in the neutral pH range and appears to be located in the cytoplasmic fraction. The purified inhibitor is sensitive to trypsin but not sensitive to neuraminidase suggesting that the inhibitor is probably a protein molecule but does not contain carbohydrate, or the carbohydrate moiety is not essential for the inhibitor activity. The inactivation of maize ADH by the inhibitor is irreversible. Furthermore, the antigenic reactivity of ADH is destroyed by the inhibitor. This result may indicate that the inhibitor is a specific proteolytic enzyme. Yeast ADH is sensitive to the inhibitor but purified horse liver ADH, maize malate dehydrogenase (MDH) and maize catalase are not affected.