{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,5,12]],"date-time":"2024-05-12T18:56:37Z","timestamp":1715540197299},"reference-count":14,"publisher":"American Physiological Society","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Regulatory, Integrative and Comparative Physiology"],"published-print":{"date-parts":[[2002,1,1]]},"abstract":"<jats:p>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 andl-[2,3-<jats:sup>3<\/jats:sup>H]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 \u00b7 mg protein<jats:sup>\u22121<\/jats:sup>\u00b7 min<jats:sup>\u22121<\/jats:sup>and 100 \u03bcM, respectively, in renal cortical homogenates from rats. The enzyme activity was the same in the presence or absence of cofactors including Ca<jats:sup>2+<\/jats:sup>, 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.<\/jats:p>","DOI":"10.1152\/ajpregu.00309.2001","type":"journal-article","created":{"date-parts":[[2015,3,3]],"date-time":"2015-03-03T20:23:30Z","timestamp":1425414210000},"page":"R303-R310","source":"Crossref","is-referenced-by-count":5,"title":["Citrulline is not the major product using the standard \u201cNOS activity\u201d assay on renal cortical homogenates"],"prefix":"10.1152","volume":"282","author":[{"given":"Kirk P.","family":"Conrad","sequence":"first","affiliation":[{"name":"Departments of Obstetrics, Gynecology, and Reproductive Sciences and of Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert W.","family":"Powers","sequence":"additional","affiliation":[{"name":"Departments of Obstetrics, Gynecology, and Reproductive Sciences and of Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alan K.","family":"Davis","sequence":"additional","affiliation":[{"name":"Departments of Obstetrics, Gynecology, and Reproductive Sciences and of Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jackie","family":"Novak","sequence":"additional","affiliation":[{"name":"Departments of Obstetrics, Gynecology, and Reproductive Sciences and of Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"24","reference":[{"key":"B1","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/S0021-9258(18)51172-8","volume":"155","author":"Blanchard M","year":"1944","journal-title":"J Biol Chem"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.87.2.682"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9673(01)84184-4"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/0165-0270(94)90126-0"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/0143-4004(95)90013-6"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1152\/ajprenal.1999.276.5.F767"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1096\/fasebj.7.13.7691671"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1172\/JCI118059"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.ph.57.030195.003423"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1042\/bj1330779"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1152\/ajprenal.1997.272.5.F561"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(74)31076-2"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-4838(97)00039-3"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.1997.6354"}],"container-title":["American Journal of Physiology-Regulatory, Integrative and Comparative Physiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.physiology.org\/doi\/pdf\/10.1152\/ajpregu.00309.2001","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,5,1]],"date-time":"2022-05-01T09:26:03Z","timestamp":1651397163000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.physiology.org\/doi\/10.1152\/ajpregu.00309.2001"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2002,1,1]]},"references-count":14,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2002,1,1]]}},"alternative-id":["10.1152\/ajpregu.00309.2001"],"URL":"https:\/\/doi.org\/10.1152\/ajpregu.00309.2001","relation":{},"ISSN":["0363-6119","1522-1490"],"issn-type":[{"value":"0363-6119","type":"print"},{"value":"1522-1490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2002,1,1]]}}}