Abstract
The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.
Highlights
The effects of the loss of MeSAdo phosphorylase on cell growth and metabolism cannot readily be ascertained using naturally enzyme-deficient cell lines that have been maintained in tissue culture for long periods of time, and may differ in muitipbways from MeSAdo phosphorylase-positive cells
We describe a novel method for the selection of MeSAdo phosphorylase-deficient mutants, and characterize two MeSAdo phosphorylase-deficient clones derived from the transplantablemurine T lymphoma cell line R1.1
The isolation and characterization of mutants is an important tool for analyzing the metabolic role of individual enzymes in intact viable mammalian cells
Summary
Selection of MeSAdo Phosphorylase-deficient Mutantsin e Partially deficient. The uptake Enzyme activities in wild type and MeSAdo phosphorylase-deficient of 5’-C1-[2,8-3H]Adoby surviving cells decreased such that mutants increasing amounts of radioactivity had tobe added to achieve Each value is the mean of at least two different measurements and a comparable amount of incorporation. The is expressed as nmol of product formed per min per mg of protein. Surviving cells no longer incorporated significant quantities of the radioactive MeSAdo analog (1x lo-’ pCi/cell)
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