Nuclear Poly(A) Polymerase from Rat Liver and a Hepatoma

Abstract

Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE Sephadex, phosphocellulose, hydroxyapatite and QAE[quaternary ammonium ethyl]-Sephadex. Purified enzyme from liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, on denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin-5''-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, .alpha.-amanitin and pancreatic RNase. The liver and hepatoma enzymes differed with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated MW (liver, 48,000; hepatoma, 60,000) and amino acid compositions. The level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.