Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids

Abstract

Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.