TY - JOUR
T1 - Characterization of O6-methylguanine-DNA-methyltransferase (O6-MGMT) activity in Xiphophorus fishes
AU - Walter, Ronald B.
AU - Sung, Huang Mo
AU - Intano, Gabe W.
AU - Walter, Christi A.
N1 - Funding Information:
This work was supported by grant awards RR-12253 from the National Center for Research Resources, CA-75137 from the National Cancer Institute, and the State of Texas Advanced Technology Program grant award ATP-030. O 6 -benzylguanine was kindly provided by the NIH-NCI Drug Synthesis Division. We also express our sincere appreciation for support from the Roy F. and Joann C. Mitte Foundation and to Steve Kazianis for critical reading of this manuscript.
PY - 2001/6/27
Y1 - 2001/6/27
N2 - We utilized a custom-synthesized double-strand oligonucleotide containing a single O6-methylguanine (O6-MG) residue within a restriction endonuclease recognition site to determine O6-methylguanine-DNA-methyltransferase (O6-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O6-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O6-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23°C. Results from protein extract activity assays indicate O6-MGMT activity patterns among four Xiphophorus tissues followed the order: brain ≥ testes > gill ≥ liver. In mammals, O6-MGMT activity is high in liver, while activity in brain is minimal (i.e. ≈9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (≈six-fold) O6-MGMT activity levels than liver. Comparison of O6-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O6-MG from the oligonucleotide substrate.
AB - We utilized a custom-synthesized double-strand oligonucleotide containing a single O6-methylguanine (O6-MG) residue within a restriction endonuclease recognition site to determine O6-methylguanine-DNA-methyltransferase (O6-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O6-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O6-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23°C. Results from protein extract activity assays indicate O6-MGMT activity patterns among four Xiphophorus tissues followed the order: brain ≥ testes > gill ≥ liver. In mammals, O6-MGMT activity is high in liver, while activity in brain is minimal (i.e. ≈9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (≈six-fold) O6-MGMT activity levels than liver. Comparison of O6-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O6-MG from the oligonucleotide substrate.
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U2 - 10.1016/S1383-5718(01)00169-3
DO - 10.1016/S1383-5718(01)00169-3
M3 - Article
C2 - 11516711
AN - SCOPUS:0035958269
SN - 1383-5718
VL - 493
SP - 11
EP - 22
JO - Mutation Research - Genetic Toxicology and Environmental Mutagenesis
JF - Mutation Research - Genetic Toxicology and Environmental Mutagenesis
IS - 1-2
ER -