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.
Conrad
Kirk P
KP
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA. rsikpc@mail.magee.edu
Powers
Robert W
RW
Davis
Alan K
AK
Novak
Jackie
J
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001120:Arginine / Q000493:pharmacokinetics; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002956:Citrulline / Q000032:analysis / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006146:Guanidines / Q000493:pharmacokinetics; D006801:Humans; D007672:Kidney Cortex / Q000201:enzymology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009249:NADP / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D051381:Rats; D020318:Rats, Long-Evans; D014316:Tritium; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000975:Antioxidants; D002250:Carbon Radioisotopes; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D006146:Guanidines; D014316:Tritium; D001708:Biopterins; D019323:omega-N-Methylarginine; D002956:Citrulline; D004533:Egtazic Acid; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester