Background: 1,25-Dihydroxyvitamin D (1,25-(OH)(2)D), the hormonal form of vitamin D, is difficult to measure because of its low circulating levels (pg/mL), and similarity to more abundant metabolites. Here a fully-automated chemiluminescent assay that accurately and precisely measures 1,25-(OH)(2)D is described.Method: The novel 1,25-(OH)(2)D assay was conceived based on four pillars: (1) the VDR's ligand binding domain (LBD) as a capture molecule; (2) reaction conditions wherein 1,25-(OH)(2)D favors binding to LBD vs. the vitamin D binding protein; (3) exploitation of liganded-LBD's conformational change; (4) a monoclonal antibody specific to liganded-LBD. This specific, conformational, sandwich approach, unique for automated measurement of haptens, is superior to more cumbersome, conventional competitive formats.Results: Accuracy of the 1,25-(OH)(2)D assay was corroborated by its alignment against LC-MS/MS with fit Deming regression equations of y = 0.98x + 1.93 (r = 0.92), and y = 1.07x + 3.77 (r = 0.94) for different methods from Endocrine Sciences, Laboratory Corporation of America (R) and the University of Washington, respectively. Good analytical precision was manifested by its low estimated limit of quantitation (1.57 pg/mL), average intra-assay imprecision (3.5%CV; range 1.1-4.7%), and average inter assay imprecision (4.5%CV; range 3.4-7.2%). Expected and measured recovery values were congruent (93.4% mean).Conclusions: The novel 1,25-(OH)(2)D method exhibited excellent correlation with well validated LC-MS/MS assays from two laboratories. Significantly, its 65 min turn-around time is quicker, and sample volume smaller (75 mu l) than current methods. (C) 2015 The Authors. Published by Elsevier Ltd.

The mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by the antibiotic L-cycloserine was investigated. L-Cycloserine is a time-dependent inactivator of GABA aminotransferase; no enzyme activity returns upon gel filtration or dialysis. Treatment of GABA aminotransferase with [C-14]-L-cycloserine, followed by rapid gel filtration, gives enzyme containing 1.1 equiv of radioactivity bound. Dialysis or denaturation by acid base, or urea releases the radioactivity. Inactivation of [H-3]pyridoxal 5'-phosphate (PLP)-reconstituted GABA aminotransferase with L-cycloserine followed by dialysis or denaturation also leads to the release of radioactivity from the enzyme. Both the released [C-14]- and [H-3]-labeled adducts comigrate by HPLC, suggesting that the inactivation adduct is a condensation product of L-cycloserine with the PLP coenzyme. By HPLC comparison, it was shown that the radiolabeled adduct is not PLP, PMP, PLP oxime, or 4-[3-hydroxy-2-methyl-5-(phosphooxymethyl)-4-pyridinyl]-2-oxo-3-butenoic acid (20), the expected product of an enamine-type inactivation mechanism. On the basis of the stability of the released adduct to acid and base and its UV-visible spectrum, which has the appearance of a PMP analogue, a simple Schiff base between PLP and cycloserine also was excluded. HPLC of the cycloserine-coenzyme adduct had a retention time very similar to that of the gabaculine-coenzyme adduct. Electrospray ionization tandem mass spectrometry of the isolated cycloserine-coenzyme adduct is consistent with a structure that is one of the tautomeric forms of the Schiff base between PMP and oxidized cycloserine (21).

L-Serine O-sulfate inactivates γ-aminobutyric acid aminotransferase (GABA-AT) by a mechanism that involves elimination of the leaving group and an enamine rearrangement, the same mechanism as that for the inactivation of GABA-AT by 4-amino-5-fluoropentanoic acid.

γ-Aminobutyric acid (GABA) aminotransferase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of GABA into succinic semialdehyde. Hydrazine analogues have long been known to act as inactivators of PLP-dependent enzymes, including GABA aminotransferase, however, no studies of the molecular mechanism of inactivation of PLP-dependent enzymes by hydrazines have been reported. 3-Hydroxybenzylhydrazine is shown to be a potent in vitro time-dependent inhibitor of pig brain GABA aminotransferase. UV-visible and 1H NMR studies, both with GABA aminotransferase and with PLP as a chemical model for the enzyme-catalyzed reaction, indicate that 3-hydroxybenzylhydrazine reacts both enzymatically and nonenzymatically to form the 3-hydroxybenzylhydrazone of PLP without tautomerization.

The mechanism of inactivation of γ-aminobutyric acid aminotransferase (GABA-AT) by l-3-chloroalanine hydroxamate (1) was investigated. Inactivation of [3H]PLP-reconstituted GABA-AT with 1 followed by denaturation gave no PMP or enamine adduct to the PLP; however, a new unknown metabolite was observed which was identical to the metabolite formed upon inactivation of GABA-AT by l-cycloserine. Time-dependent inactivation occurs, but the kinetics are second order; the rate of inactivation increases with time. After inactivation occurs the addition of fresh enzyme results in a faster rate of inactivation than prior to the initial inactivation. This indicates that the actual inactivator is generated from l-3-chloroalanine hydroxamate, and is not l-3-chloroalanine hydroxamate itself. Added gabaculine-inactivated enzyme to fresh enzyme does not increase the rate of inactivation, suggesting that the conversion of l-3-chloroalanine hydroxamate to the active form is not catalyzed by peripheral amino acid residues. l-3-Chloroalanine hydroxamate was shown to undergo buffer-catalyzed cyclization to l-cycloserine, which is the actual inactivator of GABA-AT.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanism of inactivation of .gamma.-aminobutyric acid aminotransferase by 3-hydrazinopropionic acidRichard B. Silverman and Gregory T. OlsonCite this: J. Org. Chem. 1995, 60, 19, 6232–6234Publication Date (Print):September 1, 1995Publication History Published online1 May 2002Published inissue 1 September 1995https://pubs.acs.org/doi/10.1021/jo00124a053https://doi.org/10.1021/jo00124a053research-articleACS PublicationsRequest reuse permissionsArticle Views82Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts