Gel electrophoresis and isoelectric focusing of mitochondrial and viral‐induced thymidine kinases

Abstract

AbstractSubcellular fractionation studies have shown that the thymidine (dT) kinases induced by vaccinia and herpes simplex type 1 (HSV‐1) viruses are localized principally in the cytosol fraction. Isoelectric focusing, disc polyacrylamide gel electrophoresis (disc PAGE), and glycerol gradient centrifugation experiments were carried out in order to compare the viral‐induced enzymes with cellular dT kinases from the following sources: (1) cytosol fractions of dT kinase‐positive human (HeLa S3) and mouse (LM) cells; and (2) mitochondrial extracts of HeLa S3 and of bromodeoxyuridine‐resistant, dT kinase‐deficient HeLa (BU25) and LM (TK−) cells. These analyses revealed that the sedimentation coefficients of the vaccinia and HSV‐1 induced dT kinases were similar to those of the HeLa S3 and LM cytosol enzymes, but the viral‐induced dT kinases exhibited greater disc PAGE mobilities (Rm values) and lower pI values than the HeLa S3 and LM cytosol dT kinases. The vaccinia and HSV‐1 induced dT kinases were distinctly different. Both viral‐induced enzymes also differed from LM (TK−) mitochondrial dT kinase in sedimentation coefficient, Rm and pI. Small differences were found between the HSV‐1 induced dT kinase and a human mitochondrial‐specific isozyme. However, the HSV‐1 induced dT kinase resembled the mitochondrial‐specific human and mouse dT kinases in ability to substitute GTP, UTP and CTP for ATP as phosphate donors in the dT kinase reaction. In contrast, the LM and HeLa S3 cytosol dT kinases and the vaccinia‐induced dT kinase efficiently utilized only ATP as a phosphate donor. Recovery of LM and HeLa S3 cytosol dT kinase activities from gels was very poor when ribonucleoside 5'‐triphosphates were omitted from the upper buffer solution during disc PAGE. Also, in the absence of ATP, enzyme recoveries of mitochondrial and viral‐induced dT kinases were reduced and Rm values were decreased.