Enzymatic characterisation of recombinant murine inducible nitric oxide synthase.
A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.
Moss
D W
DW
Wellcome Research Laboratories, Beckenham, Kent, UK.
Wei
X
X
Liew
F Y
FY
Moncada
S
S
Charles
I G
IG
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000502:physiology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases