Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (R _m) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 R _m, pI 9.8). The 0.6-R _m (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 R _m) enzyme and the 0.4 R _m mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-R _m activity, most of the HeLa S3 0.25-R _m activity, and some 0.4-R _m activity; (3) the inner membrane fraction is the major site of the 0.4-R _m activity but the outer membrane fraction also contains the 0.4 R _m activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-R _m dT kinase activity.