Studies on Microsomal Nucleoside Diphosphatase of Rat Hepatocytes: ITS PURIFICATION, INTRAMEMBRANOUS LOCALIZATION, AND TURNOVER

Abstract

Abstract A new procedure is described for the solubilization and purification of nucleoside diphosphatase from rat liver microsomes. The enzyme was solubilized from microsomes by an alkaline treatment (pH 10.7) in the presence of a low concentration (0.05%) of sodium deoxycholate, and purified to a nearly homogeneous state by chromatographic procedures using DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. A rabbit antiserum was prepared against the purified nucleoside diphosphatase. The antibody gave a single precipitation line with the purified enzyme as well as with the crude enzyme preparation, and strongly inhibited the nucleoside diphosphate-hydrolyzing activity of purified enzyme preparations. The nucleoside diphosphatase in intact microsomes is in a latent state, and its activity is greatly stimulated by treatments which affect the lipoprotein membrane structure of microsomal vesicles. The activation and solubilization of the enzyme by various treatments were studied in detail, and it was concluded that the enzyme is bound to the inner surface of the membrane, enclosing the microsomal vesicles. The permeability barrier of the microsomal membrane to the substrate seems to be responsible for the latency of the enzyme in intact microsomes. This conclusion was supported by the following immunochemical experiments utilizing rabbit antiserum against the purified enzyme. The enzyme in intact microsomes failed to react with the antiserum, whereas the activity of solubilized enzyme was strongly inhibited by the antiserum. When microsomes were treated with phospholipase C, the nucleoside diphosphatase became susceptible to the inhibition by the antiserum. The proteolytic digestion of intact and phospholipase C-treated microsomes also gave results which were compatible with the supposed inside localization of the nucleoside diphosphatase in microsomal vesicles. The turnover rate of the enzyme in vivo was also determined by using the specific precipitation of the microsomal nucleoside diphosphatase with the antibody. The half-life of the enzyme was 1 to 1½ days which was considerably shorter than the half-life of total microsomal protein. This finding offers additional evidence for the independent turn-over of microsomal membrane components, as suggested in several recent publications.