In vivo and in vitro expression of porcine D-amino acid oxidase: in vitro system for the synthesis of a functional enzyme.

Abstract

In vivo expression of D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, was examined by use of cloned complementary DNA [Fukui, K., Watanabe, F., Shibata, T., & Miyake, Y. (1987) Biochemistry 26, 3612-3618]. RNA blot hybridization analysis revealed that DAO is expressed abundantly in kidney and liver, is expressed significantly in brain, but is not expressed in lung of pig. Three mRNA species were expressed in kidney and liver, but only one was detected in brain. These results show the presence of tissue-specific regulation of DAO gene expression. In vitro expression of a functional enzyme was achieved through the construction of a recombinant plasmid containing an SP6 promoter and a restriction enzyme fragment of cDNA to generate a DAO-specific RNA transcript. The in vitro translation product of the capped RNA transcript showed significant catalytic activity, which was inhibited strongly by benzoate, a potent inhibitor of DAO. The kinetic properties of the in vitro synthesized enzyme were comparable to those of the purified enzyme from porcine kidney. It is now possible to synthesize a functional D-amino acid oxidase in vitro and to investigate its structure-function relationships.