Enzyme kinetics of N-nitrosodimethylamine demethylase in rodents and humans.
A variety of Km values have been reported for hepatic microsomal N-nitrosodimethylamine demethylase (NDMAd). We demonstrated previously that the biologically important, high affinity (KmI) form of microsomal NDMAd is manifested by cytochrome P450IIE1 (also known as P450ac and P450j). The KmI value of NDMAd was, however, affected greatly by assay conditions: the possible presence of inhibitors and the presence of cytochrome b5. We re-examined the KmI value by testing the effect of enzyme concentrations and of different types of enzyme preparations on the Km. The KmI value ranged from 15 to 22 microM, as estimated by the direct linear plot, using a microsomal protein concentration in the range of 0.1 to 0.8 mg/ml with correction for substrate utilization. A slight yet significant dependency of microsomal protein concentration on the Km (r = 0890; p less than 0.05) was seen. When five different microsomal preparations were compared, the KmI value ranged from 14 to 24 microM (median, 20 microM), as estimated by the direct linear plot. The Km estimated by the commonly used Eadie-Hofstee plot did not differ from that by the direct linear plot. These Km values are close to the values obtained in studies with isolated cells and tissue slices. The KmI form of NDMAd (P450IIE1 and its orthologues) is present in rats, mice, rabbits, hamsters and guinea-pigs. It is responsible for the age-dependent differences between rats and hamsters and for the sex-related differences in mouse kidneys, and for the bioactivation and toxicity of NDMA. This enzyme also exists in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoo
J S
JS
College of Pharmacy, Rutgers University, Piscataway, NJ.
Ishizaki
H
H
Yang
C S
CS
D000818:Animals; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D004128:Dimethylnitrosamine