Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

Journal Article · Tue Jan 05 04:00:00 EST 1988 · J. Biol. Chem.; (United States)

OSTI ID:7075801

Sherley, J L; Kelly, T J

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, the authors 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/sub 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 A 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/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

• Research Organization: Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA)
• OSTI ID: 7075801
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:1; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
CELL CYCLE
DNA REPLICATION
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
FILTRATION
HELA CELLS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MOLECULAR WEIGHT
NUCLEIC ACID REPLICATION
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PURIFICATION
SEDIMENTATION
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS