Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells

J. L. Sherley, Thomas Kelly

Research output: Contribution to journalArticle

Abstract

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, we have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R(m) of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 Å determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M(r) = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity. 1) The M(r) = 24,000 polypeptide co-migrates with thymidine kinase activity in electrophoretic and sedimentation analyses. 2) A subunit of M(r) = 25,504 is predicted by the nucleotide sequence of a recently isolated cDNA clone that encodes HeLa thymidine kinase. 3) Mouse LTK- cells transformed with this clone express a cytosolic thymidine kinase activity, as well as a novel M(r) = 24,000 polypeptide detectable with immune serum raised against the purified human enzyme.

Original languageEnglish (US)
Pages (from-to)375-382
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number1
StatePublished - 1988

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Thymidine Kinase
HeLa Cells
Purification
Enzymes
Sedimentation
S Phase
Peptides
Immune Sera
Gels
Clone Cells
Affinity chromatography
Digitonin
Enzyme activity
Ion Exchange Chromatography
Methotrexate
Sodium Dodecyl Sulfate
Glycerol
Hydrodynamics
DNA Replication
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

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Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells. / Sherley, J. L.; Kelly, Thomas.

In: Journal of Biological Chemistry, Vol. 263, No. 1, 1988, p. 375-382.

Research output: Contribution to journalArticle

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