PubMed · 8700218
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S Kauffman. 1996-08-08. Self-replication. Even peptides do it.. https://doi.org/10.1038/382496a0
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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.
Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl damages proteins by reaction with amino acid side-chains or backbone cleavage. Little information is available about the mechanisms and intermediates involved in these reactions. EPR spin trapping has been employed to identify radicals on proteins, peptides and amino acids after treatment with HOCl. Reaction with HOCl gives both high- and low-molecular-mass nitrogen-centred, protein-derived radicals; the yield of the latter increases with both higher HOCl:protein ratios and enzymic digestion. These radicals, which arise from lysine side-chain amino groups, react with ascorbate, glutathione and Trolox. Reaction of HOCl-treated proteins with excess methionine eliminates radical formation, which is consistent with lysine-derived chloramines (via homolysis of N-Cl bonds) being the radical source. Incubation of HOCl-treated proteins, after removal of excess oxidant, gives rise to both nitrogen-centred radicals, over a period of hours, and time-dependent fragmentation of the protein. Treatment with excess methionine or antioxidants (Trolox, ascorbate, glutathione) protects against fragmentation; urate and bilirubin do not. Chloramine formation and nitrogen-centred radicals are therefore key species in HOCl-induced protein fragmentation.
1. The intestinal transport of L-methionine has been investigated in brush-border membrane vesicles isolated from the jejunum of 6-week-old chickens. L-Methionine influx is mediated by passive diffusion and by Na+-dependent and Na+-independent carrier-mediated mechanisms. 2. In the absence of Na+, cis-inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L-methionine influx: one sensitive to L-lysine and the other sensitive to 2-aminobicyclo[2.2. 1]heptane-2-carboxylic acid (BCH). The L-lysine-sensitive flux is strongly inhibited by L-phenylalanine and can be broken down into two pathways, one sensitive to N-ethylmaleimide (NEM) and the other to L-glutamine and L-cystine. 3. The kinetics of L-methionine influx in Na+-free conditions is described by a model involving three transport systems, here called a, b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km = 2.2 +/- 0.3 microM and Vmax = 0.13 +/- 0.005 pmol (mg protein)-1 (2 s)-1; system b: Km = 3.0 +/- 0.3 mM and Vmax = 465 +/- 4.3 pmol (mg protein)-1 (2 s)-1); system c is specific for neutral amino acids, has a Km of 1.29 +/- 0.08 mM and a Vmax of 229 +/- 5.0 pmol (mg protein)-1 (2 s)-1 and is sensitive to BCH inhibition. 4. The Na+-dependent component can be inhibited by BCH and L-phenylalanine but cannot interact either with cationic amino acids or with alpha-(methylamino)isobutyrate (MeAIB). 5. The kinetic analysis of L-methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3-fold decrease in the Michaelis constant and a 1.4-fold increase in Vmax. In the presence of Na+, the BCH-sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 +/- 0. 013 mM and a Vmax of 391 +/- 2.3 pmol (mg protein)-1 (2 s)-1. 6. It is concluded that L-methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L-methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.