PubMed · 282190
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1978. [Artificial sweeteners and their problems].. https://pubmed.ncbi.nlm.nih.gov/282190/
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Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.
The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.
The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine-2,4- dinitrophenyl, where P(2), P(2)', and P(3) residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P(1) and P(1)' residues, and adjacent residues are designated P(2), P(3), P(2)', P(3)', etc.) There was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P(2) residues having the greatest effect. The S(3), S(2), and S(2)' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P(2) position, were among the best P(2)' residues. The specificity at P(3) was generally opposite that of P(2). Residues at P(2), and to a lesser extent at P(3), influenced the cleavage site. At the P(2) position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P(2) Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P(1)' or P(1) position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.