The Role of Neurotensin in Control of Anterior Pituitary Hormone Secretion
Craig A. Johnston, Charles D. Barnes in Brain-Gut Peptides and Reproductive Function, 2020
While attempting to purify corticotropin-releasing factor from hypothalamic extracts, Susan Leeman noticed that the rats salivated following injection of certain fractions (Leeman and Carraway, 1982). She and her associates characterized the molecule responsible for this sialogogic activity. It was substance P (Chang and Leeman, 1970). While screening columns to locate sialogogic activity, she noticed a characteristic vasodilatation of exposed cutaneous regions, particularly around the face and ears of the assay rats. She and her associates went on to isolate and determine the structure of this vasodilatory peptide which was named neurotensin (Carraway and Leeman, 1973). The structure of the tridecapeptide (Fig. 1) has pyroglutamic acid at the N-terminal and a carboxyl group at the C-terminal end of the molecule.
Liquid Phase Sequence Analysis of Proteins/Peptides
Ajit S. Bhown in Protein/Peptide Sequence Analysis: Current Methodologies, 1988
Following coupling, the next reaction in stepwise degradation is the cleavage of the PTC-peptide, which is achieved by lowering the pH for a short period of time. Fluoroacids, such as heptofluorobutyric acid (HFBA) or trifluoroacetic acid (TFA), are the most commonly employed acids, because they are excellent solvents for proteins and cause few side reactions.22 The cleavage reaction is an equilibrium reaction rather than a quantitative reaction; however, the equilibrium is close to being quantitative.32 Cleavage is dependent upon the type of amino acid being cleaved and the amino acid adjacent to the one being cleaved.35 When proline is the amino acid being cleaved, introduction of double cleavage followed by extraction after each cleavage and collection into the same tube improves the proline recovery.31,35,37 Double cleavage has been proposed for aspartic acid and glutamic acid,22 while shorter cleavage time for glutamine helps to avoid the possibility of cyclization to pyroglutamic acid.32 The resultant ATZ derivatives of amino acids are unstable and for this reason are converted to the more stable phenylthiohydantoin (PTH) derivatives.
Kappa III Immunoglobulin Light Chain Origin of Localized Orbital Amyloidosis
Gilles Grateau, Robert A. Kyle, Martha Skinner in Amyloid and Amyloidosis, 2004
Chromatography of guanidine solubilized fibrils on Sepharose CL6B yielded two retarded peaks eluting in the approximately 10kD and ≤ 5kD areas. SDS-PAGE analysis of the higher molecular weight pool showed a band at approximately 10kD, and of the lower molecular weight pool a broad smear in the 3-5kD region. Sequence analysis of the 10kD pool showed a major sequence starting with residue 150 of the constant region of an immunoglobulin kappa light chain. Analysis of the lower molecular weight pool indicated the presence of numerous peptides, some of which started with residues 127 and 150 of kappa constant region. Analysis of tryptic peptides from the 10kD pool identified residues 3-61 of kappa light chain variable region as the major constituents (Figure 1). Peptides from residues 150-207 of kappa constant region were present, but in approximately 20-25% the molar amounts of variable region. The 50% acetic acid insoluble pellet from the trypsin digest contained a peptide starting with residue 62 and continuing to residue 99 but in less than 5% the molar amount of other variable region tryptic peptides. Analysis of the 10kD pool after pyroglutamate aminopeptidase digestion showed a new sequence starting with Arg2 of the variable region and identified the N-terminus as pyroglutamic acid.
Liver metabolomic characterization of Sophora flavescens alcohol extract-induced hepatotoxicity in rats through UPLC/LTQ-Orbitrap mass spectrometry
Published in Xenobiotica, 2020
Peng Jiang, Yancai Sun, Nengneng Cheng
Glutathione is an essential cellular antioxidant synthesized from glutamine carbons. Glutathione can prevent cellular damage caused by alkylating agents and free radicals by binding to electrophilic chemicals. Glutathione, especially in the liver, binds to toxic chemicals to detoxify them. Pyroglutamic acid is a cyclized intermediate in the γ-glutamyl cycle, a pathway for glutathione biosynthesis and degradation. This intermediate is also related to redox imbalance. Increased levels of pyroglutamic acid and glutathione might reflect the hepatotoxicity of SFAE associated with the disturbance of glutathione metabolism.
Preformulation studies of l -glutathione: physicochemical properties, degradation kinetics, and in vitro cytotoxicity investigations
Published in Drug Development and Industrial Pharmacy, 2020
Mengyang Liu, Manisha Sharma, Guo-Liang Lu, Naibo Yin, Murad Al Gailani, Sree Sreebhavan, Jingyuan Wen
The unknown peaks (peak 1*, 2*, 3*, 4*, 5*, 6*, and 7*) observed in HPLC analysis of GSH degradation were identified by high resolution LC-MS. The seven mass peaks shown in Figure 11 were detected and attributed to GSSG (a: peak 1*), cysteinyl glycine (b: peak 2*), pyroglutamic acid (c: peak 3*), glutathione (d: drug main peak), glutamic acid (e: peak 4*), glutathione sulfinate (f: peak 5*), gamma-glutamylcysteine dioxide (g: peak 6*) and glutathione sulfonic acid (h: peak 7*). It was observed that in acidic conditions, the main degradation products were glutathione disulfide, cysteinyl glycine, glutathione, pyroglutamic acid, glutamic acid, and to a lesser extent GSSG. These (acidic) degradation products indicate amide bond cleavage between glutamate and cysteine, which was further acid-catalyzed into pyroglutamic acid. Under basic conditions there was also amide bond cleavage between glutamate and cysteine but also the amide bond between cysteine and glycine was cleaved to form glutamic cysteine sulfonate, which was further base-catalyzed into gamma-glutamylcysteine dioxide (peak 6*). A small amount of GSH was also oxidized into glutathione sulfinate in basic conditions. Expectedly, in the presence of pure water and artificial light only the formation of GSSG by oxidation was observed without any amide bond cleavage due to its autoxidation. Exposing GSH to high temperatures would result in amide bond breakage, but only between glutamate and cysteine. Treatment of GSH with hydrogen peroxide leads to the formation of GSSG (peak 1*) with a small amount of glutathione sulfonic acid (peak 7*). In summary, a schematic degradation scheme of GSH in different conditions is shown in Figure 12. The different degradation mechanisms and kinetics corresponded to a specific degradation kinetic model analysis (two kinetic orders existed in six different conditions).
Effects of N-terminal and C-terminal modification on cytotoxicity and cellular uptake of amphiphilic cell penetrating peptides
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Mehdi Soleymani-Goloujeh, Ali Nokhodchi, Mehri Niazi, Saeedeh Najafi-Hajivar, Javid Shahbazi-Mojarrad, Nosratollah Zarghami, Parvin Zakeri-Milani, Ali Mohammadi, Mohammad Karimi, Hadi Valizadeh
An old well-known method to modify a peptide sequence or protein motif is N- and C-terminal modification to fulfil some ideas like resistance against exo-peptidases cleavage. N-terminal modification of peptides was done with 5(6)-FAM in the case of uptake studies. On the other hand, pyroglutamic acid was chosen to N-terminal modification of another group of peptides to use in cytotoxicity and cell viability assays. The amino protecting groups were removed by piperidine/DMF 20–40% Solution (2 ml). The resin was washed with DMF, then rinsed with DCM (3 × 2 ml) to remove DMF and dried by a vacuum pump. The desired amount of peptide-resin was weighed and poured into a reaction vessel. In a separate vessel, FAM was dissolved in DMF, then TBTU and DIPEA were added and mixed gently. The solution was added to slurry the resin and vessel was stirred for three hours at room temperature. A Kaiser test was performed to confirm end point. The resin was washed with DMF (3 × 4 ml), isopropanol (3 × 4 ml) and DCM (3 × 4 ml). On the other hand, pyroglutamic acid (pyrrolidone carboxylic acid; pGlu) was found as the N-terminal amino acid in various proteins and polypeptides. N-terminal modification with pyroglutamic acid was performed in cytotoxicity and cell viability assays. The coupling procedure was as same as FAM labeling procedure. C-terminal modification was done by applying memantine (Mem) in sequences which were used in uptake studies, also benzylamine (BA) was used in cytotoxicity and cell viability assays. Based on the dried amount of peptide-resin, the produced N-acetylated peptides were cleaved from the resin by treatmenting TFA in DCM (1–2%) with the final volume of 5 ml and each reaction vessel was stirred two hours at room temperature. Neutralization process was performed by calculating the amount of excess DIPEA, then C-amidation of the synthesized peptides was carried out using Mem or BA and HOBt in the presence of TBTU as a coupling reagent, using DIPEA as the base and DMF/DCM mixture as solvent at room temperature. The mixture in a three-neck glass flask was stirred by a magnetic stirrer overnight under vacuum.
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