PubMed · 4370908
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L H Jensen. 1974. Protein model refinement based on x-ray data.. https://doi.org/10.1146/annurev.bb.03.060174.000501
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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.
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.
A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding-decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED50 values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the gamma-carbon. The excellent correlation between these relative ED50 values and a series of relative Kd values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.