Mammalian DNA ligase. Structure and function in rat-liver tissues

Abstract

DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed, leading to the isolation of one or two peaks (fractions A and B) on DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4.degree. C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.