Inactive O6-methylguanine-DNA methyltransferase in human cells.
A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.
Zhukovskaya
N
N
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Rydberg
B
B
Karran
P
P
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics
C059417:N protein, Bacteriophage lambda; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase