Studies on Rat Liver Catalase VIII. Isolation and Translation of Catalase Messenger RNA*

Messenger RNA directing the biosynthesis of catalase [EC 1.11.1.6] has been purified from rat liver. The procedure consists of: (1) isolation of catalase-synthesizing polysomes by immunoprecipitation, (2) extraction of RNA from the polysomes with phenol, and (3) adsorption of mRNA on nitrocellulose filters. Isolated catalase mRNA was translated by a cell-free system using ribosomes prepared from eggs of a brine shrimp, Artemia salina, and cell sap of rat liver. In this system the mRNA directed the incorporation of uC-leucine into catalase at least 70 % as efficiently as into total proteins. Catalase mRNA was obtained from both free and membrane-bound polysomes. The main component in the purified catalase mRNA preparation was pulse-labeled with [3H]orotic acid, and was assumed to be about 15S on the basis of sucrose gradient centrifugation. Nitrocellulose filters were effective in the purification of catalase mRNA, suggesting that this messenger contains a poly (A) segment, as is the case in some other mammalian messengers. The procedure for isolating natural messengers which direct the biosynthesis of specific pro-teins consists of: (1) separation of polysomes synthesizing a specific protein, (2) extraction of RNA from such polysomes, and (3) purifi-cation of mRNA for the protein. In the course of studies on the biosynthesis of rat liver cata-lase [EC 1.11.1.6], Higashi et al. (7, 2) have established a method for isolating catalase-synthesizing polysomes, based on the immuno-chemical reaction between nascent catalase on the polysomes and anti-catalase antibodies. O