Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barath, Z., and Küntzel, H. (1972). Induction of mitochondrial RNA polymerase in Neurospora crassa. Nature New Biol. 240195.
Berk, A. J., and Clayton, D. A. (1973). A genetically distinct thymidine kinase in mammalian mitochondria: Exclusive labelling of mitochondrial deoxyribonucleic acid. J. Biol. Chem. 2482722.
Berk, A. J., Meyer, B. J., and Clayton, D. A. (1973). Mitochondrial-specific thymidine kinase. Arch. Biochem. Biophys. 154563.
Dubbs, D. R., Kit, S., deTorres, R. A., and Anken, M. (1967). Virogenic properties of bromodeoxyuridine-sensitive and bromodeoxyuridine-resistant simian virus 40-transformed mouse kidney cells. J. Virol. 1968.
Kit, S., and Leung, W.-C. (1974). J. Cell. Biol. 61.
Kit, S., and Minekawa, Y. (1972). Mitochondrial thymidine-deoxyuridine-phosphorylating activity and the replication of mitochondrial DNA. Cancer Res. 322277.
Kit, S., Dubbs, D. R., Piekarski, L. J., and Hsu, T. C. (1963). Deletion of thymidine kinase activity from L-cells resistant to bromodeoxyuridine. Exptl. Cell. Res. 31297.
Kit, S., Dubbs, D. R., and Fearson, P. M. (1966). HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity. Internat. J. Cancer 119.
Kit, S., Nakajima, K., Kurimura, T., Dubbs, D. R., and Cassingena, R. (1970). Monkeymouse hybrid cell line containing the SV40 genome in a partially repressed state. Internat. J. Cancer 51.
Kit, S., Kaplan, L. A., Leung, W.-C., and Trkula, D. (1972). Mitochondrial thymidine kinase of bromodeoxyuridine-resistant, kinase-deficient HeLa (BU25) cells. Biochem. Biophys. Res. Commun. 491561.
Kit, S., Leung, W.-C., and Trkula, D. (1973a). Properties of thymidine kinase enzymes isolated from mitochondrial and cytosol fractions of normal, bromodeoxyuridine-resistant, and virus-infected cells. In Mehlman, M. A., and Hanson, R. W. (eds.), Symposium on Metabolic Regulation, New York (in press).
Kit, S., Leung, W.-C., and Kaplan, L. A. (1973b). Distinctive molecular forms of thymidine kinase in mitochondria of normal and bromodeoxyuridine resistant HeLa cells. Europ. J. Biochem. 3943.
Kit, S., Leung, W.-C., and Trkula, D. (1973c). Properties of mitochondrial thymidine kinases of parental and enzyme-deficient HeLa cells. Arch. Biochem. Biophys. 158503.
Kit, S., Leung, W.-C., and Trkula, D. (1973d) Distinctive properties of mitochondrial thymidine (dT) kinase from bromodeoxyuridine-resistant mouse lines. Biochem. Biophys. Res. Commun. 54455.
Kit, S., Leung, W.-C., Trkula, D., and Jorgensen, G. (1974). Gel electrophoresis and isoelectric focusing of mitochondrial and viral-induced thymidine kinases. Int. J. Cancer 13.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193265.
Righetti, P., and Drysdale, J. W. (1971). Isoelectric focusing in polyacrylamide gels. Biochim. Biophys. Acta 23617.
Schnaitman, C., and Greenawalt, J. W. (1968). Enzymatic properties of the inner and outer mitochondrial membranes of rat liver mitochondria. J. Cell Biol. 38158.
Shows, T. B. (1972). Genetics of human-mouse somatic cell hybrids: Linkage of human genes for lactate dehydrogenase-A and esterase-A4. Proc. Natl. Acad. Sci. 69348.
Stafford, M. A., and Jones, O. W. (1972). The presence of “fetal” thymidine kinases in human tumors. Biochim. Biophys. Acta 277439.
Author information
Authors and Affiliations
Additional information
This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-11, CA-10893, and 1-K6-AI-2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.
Rights and permissions
About this article
Cite this article
Kit, S., Leung, WC. Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells. Biochem Genet 11, 231–247 (1974). https://doi.org/10.1007/BF00486058
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00486058