Citrulline is not the major product using the standard "NOS activity" assay on renal cortical homogenates.
A standard approach to assessing nitric oxide synthase (NOS) activity in tissue homogenates is 1) removal of small-molecular-weight substances by size-exclusion chromatography, 2) adding back of substrates/cofactors in precise concentrations with a radioactive isotope of arginine (Arg), and 3) quantification of labeled citrulline (Cit) after separation of Arg and Cit ... by cation-exchange column chromatography. Using this approach and L-[2,3-3H]Arg, we found that the major product(s) was not Cit in cortical homogenates prepared from rat, mouse, and human kidneys. The product(s) mimicked Cit, insofar as it passed freely through cation-exchange columns and comigrated with Cit on both one-dimensional and two-dimensional straight-phase thin-layer chromatography systems. However, it was clearly resolved from Cit by precolumn derivatization and reverse-phase HPLC. The maximum velocity and Michaelis-Menten constant were approximately 100 pmol x mg protein(-1) x min(-1) and 100 microM, respectively, in renal cortical homogenates from rats. The enzyme activity was the same in the presence or absence of cofactors including Ca2+, calmodulin, tetrahydrobiopterin, and NADPH. It was only modestly inhibited by L-Arg analogs and was mainly in the supernatant after a 100,000 g centrifugation. These enzyme characteristics contrasted markedly with those simultaneously obtained for NOS activity in placental homogenates. Thus results from the conventional NOS activity assay should be viewed cautiously.
Mesh Terms:
Animals, Antioxidants, Arginine, Biopterin, Calcium, Carbon Radioisotopes, Chelating Agents, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, Citrulline, Egtazic Acid, Enzyme Inhibitors, Female, Guanidines, Humans, Kidney Cortex, Male, Mice, Mice, Inbred Strains, NADP, NG-Nitroarginine Methyl Ester, Nitric Oxide Synthase, Placenta, Rats, Rats, Long-Evans, Tritium, omega-N-Methylarginine
Animals, Antioxidants, Arginine, Biopterin, Calcium, Carbon Radioisotopes, Chelating Agents, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, Citrulline, Egtazic Acid, Enzyme Inhibitors, Female, Guanidines, Humans, Kidney Cortex, Male, Mice, Mice, Inbred Strains, NADP, NG-Nitroarginine Methyl Ester, Nitric Oxide Synthase, Placenta, Rats, Rats, Long-Evans, Tritium, omega-N-Methylarginine
Am. J. Physiol. Regul. Integr. Comp. Physiol.
Date: Jan. 01, 2002
PubMed ID: 11742852
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