Identification of a high-molecular-weight nonfunctional protein in L1210 leukemia cells with common antigenic determinants to dihydrofolate reductase.
The concentration of immunoreactive protein in the cytosol of L1210 cells measured using a specific radioimmunoassay for dihydrofolate reductase was substantially greater than the concentration of active enzyme which was measured by the binding of [3H]methotrexate. When the cytosol was subjected to gel filtration, two immunoreactive proteins were separated, a high-molecular-weight (Mr 318,000) protein which did not have catalytic activity and which did not bind [3H]methotrexate and a smaller protein (Mr approximately 20,000) which did reduce [3H]folic acid to tetrahydrofolate and did bind [3H]methotrexate. The nonfunctional high-molecular-weight protein neutralized the inhibitory effect of the antiserum on active dihydrofolate reductase. There was no spontaneous disaggregation of the big species into smaller subunits nor did 8 M urea alone, dithioerythritol alone, boiling with a mixture of 8 M urea and dithioerythritol, or RNase alter its apparent molecular weight. Trypsin, however, digested both the nonfunctional and active immunoreactive forms of the enzyme. Isoelectric focusing of the cytosol separated two nonfunctional immunoreactive isoproteins, each having the same isoelectric points as the two active isoenzymes of dihydrofolate reductase (pls of 8.0 and 8.5). Studies in rapidly replicating and stationary-phase L1210 cells showed that the concentration of the nonfunctional immunoreactive protein increased rapidly, reaching a peak on Day 2 of log growth at which time active enzyme was at a nadir, and then decreased rapidly, reaching a nadir on Day 4, at which time active enzyme was at a peak. The identical isoelectric points for the inactive and active immunoreactive proteins and the reciprocal concentration of each form in logarithmically growing cells suggest that the immunoreactive large species may be a precursor of the active enzyme.
D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D003600:Cytosol / Q000201:enzymology; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera; D007939:Leukemia L1210 / Q000201:enzymology / Q000276:immunology*; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D011485:Protein Binding; D011863:Radioimmunoassay; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification*
D000951:Antigens, Neoplasm; D000939:Epitopes; D007106:Immune Sera; D009363:Neoplasm Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate