Relation between cell cycle stage and the activity of DNA-synthesizing enzymes in cultured human lymphoblasts: investigations on cell fractions enriched according to cell cycle stages by way of centrifugal elutriation.
Undifferentiated human lymphoblasts (culture LS-2) were separated according to cell size during their exponential growth phase by way of centrifugal elutriation. The cell fractions thus obtained were characterized in terms of different cell cycle stages by flow cytometric measurement of their deoxyribonucleic acid (DNA histogram), the [3H]thymidine labeling index, and by determining the rate of [3H]thymidine incorporation. In these cell fractions the activities of thymidine kinase, thymidylate synthase, DNA polymerase, dihydrofolate reductase, methionine synthase, and hexokinase were determined. The results showed that all the enzymes investigated exhibited activities in all cell fractions. With the exception of DNA polymerase, all of the enzymes exhibited the lowest level of activity in the fraction containing the highest proportion of G0 + G1 phase cells (fraction 2); the activity of thymidine kinase was particularly low. This would suggest that thymidine kinase is not active in G0 + G1 phase cells and that the activity measured in fraction 2 is perhaps attributable to contamination of this fraction by S and G2 + M phase cells.
Pelka-Fleischer
R
R
Medizinische Klinik III, Klinikum Grosshadern der Ludwig-Maximilians-Universität München, Federal Republic of Germany.
Ruppelt
W
W
Wilmanns
W
W
Sauer
H
H
Schalhorn
A
A
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000378:metabolism; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000201:enzymology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D013940:Thymidylate Synthase; D006593:Hexokinase; D013937:Thymidine Kinase; D004259:DNA-Directed DNA Polymerase