Studies on Rat Liver Catalase: I. COMBINED IMMUNOCHEMICAL AND ENZYMATIC DETERMINATION OF CATALASE IN LIVER CELL FRACTIONS

Abstract

Enzymatic activity measurements give incomplete information on the distribution of an enzyme among cell components. By combining an enzymatic measurement with an immunochemical assay, the existence of inactive forms of the enzyme, or of inhibitors or activators, can frequently be detected. Use of a precipitin procedure has the added advantage that the protein is isolated as it is assayed, and incorporation of labeled substrates can be followed. The distribution of catalase in rat liver cell fractions in terms of enzymatic activity has been reported by Von Euler and Heller (I), Ludewig and Chanutin (2)) Greenfield and Price (3), Thomson and Klipfel (4), de Duve et al. (5), and Fourcade and Rosenberg (6). The immunochemical approach has been employed with bovine liver (7-g)) mouse liver (lo), and rat liver (11). However, immunochemical studies have not as yet been applied to isolated cell fractions. In a previous application of the immunochemical method to rat liver, Higashi, Yagi, and Hirai (11) employed an antiserum against human red blood cell catalase which precipitated rat liver catalase to some extent. In the present work a purified preparation of rat liver catalase has been obtained as antigen. Large scale preparations of liver mitochondria were used as the starting material for the catalase purification, since it was desired to avoid possible heterogeneity due to the existence of multiple forms of catalase within the cell. For preparation of liver cell fractions a version of the de Duve procedure (12) was selected, which permitted complete recovery of fairly pure components and also yielded a lysosome-rich fraction. Separate preparations were also made of “rough surfaced” and “smooth surfaced” microsomes and purified nuclei. The distribution of catalase in these cell fractions was ascertained by both the enzymatic and immunochemical methods. Values obtained by the two methods were in good agreement except in microsomes, for which the immunochemical result was 2 to 3 times the enzymatic one.