Penetration of mouse fibroblasts by 2′-deoxyadenosine 5′-phosphate and incorporation of the nucleotide into DNA

✍ Scribed by William Plunkett; Seymour S. Cohen

• Publisher: John Wiley and Sons
• Year: 1977
• Tongue: English
• Weight: 742 KB
• Volume: 91
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1040910211

✦ Synopsis

Abstract

The metabolism of 2′‐deoxyadenosine 5′‐phosphate (dAMP) by exponentially growing mouse fibroblasts (L‐cells) in suspension culture has been studied. Cells incubated for four hours with 0.10 μmole [^3^H, ^32^P]dAMP/ml in medium containing 9.6 μmole ^31^P~i~/ml incorporate both activities at nearly linear rates into acid‐soluble and acid‐insoluble fractions. The ^3^H/^32^P value increased about 30‐fold in each fraction during the incubation, indicating extensive dephosphorylation of dAMP. The DNA from treated cells was degraded enzymatically to 5′‐mono‐nucleotides, which were fractionated by ion‐exchange chromatography. ^3^H was associated exclusively with dAMP (53%) and dGMP (47%). ^32^P, associated with all deoxynucleotides, was at a 20‐fold higher specific activity in dAMP than in either dGMP or dCMP. The specific activity of [^32^P]dAMP incorporated into DNA in four hours was 24‐fold greater than that of ^32^P~i~ in the cellular pool. In experiments in which cells were incubated with ^32^P~i~ plus or minus 0.10 μmole dAMP/ml, the specific activity of dAMP was slightly less than that of dGMP or dCMP, which were equal. These results suggest that the higher specific activity of [^32^P]dAMP in the DNA of cells after incubation with doubly‐labeled dAMP was due to the intact penetration of some dAMP into the cells with its subsequent incorporation into DNA. Calculations, based on the amount of exogenously added [^3^H]thymidine incorporated into the cellular DNA in parallel cultures and the ^32^P of dAMP isolated from this DNA, suggest that 1% of the total dAMP residues incorporated during the 4‐hour incubation were derived directly from intact, exogenously added dAMP.