TY - JOUR
T1 - Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells
AU - Sherley, J. L.
AU - Kelly, T. J.
PY - 1988
Y1 - 1988
N2 - 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.
AB - 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.
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M3 - Article
C2 - 3335503
AN - SCOPUS:0023879292
SN - 0021-9258
VL - 263
SP - 375
EP - 382
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -