Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

1985 ◽  
Vol 5 (10-11) ◽  
pp. 847-854 ◽  
Author(s):  
Christopher C. Q. Chin ◽  
Finn Wold
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Coho Salmon ◽  
Standard Procedure ◽  
Exchange Chromatography ◽  
Rabbit Muscle ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Acylated Proteins

A standard procedure for the identification of the N-terminal amino acid in Nα-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked α-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acytase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl- and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, Nα-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1n HCl at 110° for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.

Effect of Deproteinating Agents (Picric Acid and Sulfosalicylic Acid) on Analysis for Free Amino Acids in Swine Blood and Tissue

1969 ◽  
Vol 52 (5) ◽  
pp. 981-984 ◽  
Author(s):  
J E Knipfel ◽  
D A Christensen ◽  
B D Owen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino ◽  
Biological Fluids ◽  
Picric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sulfosalicylic Acid ◽  
Coefficients Of Variation

Abstract Amino acid analyses were performed on samples of blood, liver tissue, loin muscle, and ham muscle by ion exchange chromatography after deproteination of the samples with picric acid or sulfosalicylic acid (SSA). Resolution of threonine and serine from the ion exchange column was poor when SSA was used as the deproteinating agent. Twelve of sixteen amino acids were higher (P < 0.05) in serum deproteinated with picric acid as compared to concentrations determined after SSA deproteination. Amino acid values for ham muscle tended to be higher after deproteination with picric acid; however, with liver and loin muscle samples, the values were somewhat higher after SSA deproteination. In both serum and tissue analyses, coefficients of variation were lower for niGSt amino acids when picric acid was utilized as the deproteinating agent. The latter observation, in particular, suggests that picric acid is preferable to SSA as a deproteinating agent before amino acid analyses of biological fluids. Standardization of methods of deproteination is needed to allow meaningful comparisons of data.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

scholarly journals Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization

1990 ◽  
Vol 269 (1) ◽  
pp. 85-91 ◽  
Author(s):  
J F Sinclair ◽  
S Wood ◽  
L Lambrecht ◽  
N Gorman ◽  
L Mende-Mueller ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Chicken Liver ◽  
Terminal Amino Acid ◽  
Catalytic Activities ◽  
Terminal Amino ◽  
Cytochrome P 450 ◽  
Terminal Amino Acid Sequence

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.

Nuclear Magnetic Resonance Studies of Phenylthiohydantoin Amino Acids

1975 ◽  
Vol 53 (9) ◽  
pp. 1005-1009 ◽  
Author(s):  
C. S. Tsai ◽  
N. L. Fraser ◽  
H. Avdovich ◽  
J. P. Farant
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Magnetic Resonance ◽  
Diagnostic Purpose ◽  
Edman Degradation ◽  
Terminal Amino Acid ◽  
Magnetic Resonance Studies ◽  
Terminal Amino ◽  
Derivatives Of

Proton magnetic spectra of 3-phenyl-2-thiohydantoin derivatives of common amino acids in deuterated dimethyl sulfoxide were recorded. Spectral data pertaining to characteristic protons for diagnostic purpose were compiled. Their application to the N-terminal amino acid analysis of peptide by Edman degradation was examined.

scholarly journals Phosphorus (V) porphyrin diaxially substituted with Leu-enkephalin

2004 ◽  
Vol 2 (3) ◽  
pp. 446-455 ◽  
Author(s):  
Mariusz Gajewski ◽  
Leszek Czuchajowski
Keyword(s):  
Amino Acids ◽  
Photodynamic Therapy ◽  
Amino Acid ◽  
Biologically Active ◽  
Solution Phase ◽  
Terminal Amino Acid ◽  
Leucine Enkephalin ◽  
Potential Applications ◽  
Biologically Active Peptide ◽  
Terminal Amino

AbstractSynthesis of the first phosphorus (V) porphyrin-peptide conjugate was successfully accomplished. A biologically active peptide, leucine enkephalin, was constructed on the phosphorus atom of the 5,10,15,20-meso-tetraphenylporphinato dichlorophosphorus (V) chloride. The method involved solution phase peptide synthesis. The first C-terminal amino acid in the sequence of the peptide was axially attached to the porphyrin through a linker, 3-aminopropanol, and the remainder of leucine enkephalin was synthesized by subsequent additions of amino acids. Leucine enkephalin-P(V) porphyrin conjugate represents a new group of compounds, and its synthesis broadens potential applications of P(V) porphyrine, e.g. in photodynamic therapy.

Ion exchange chromatography of 2-amino-2-deoxy-D-hexoses

1970 ◽  
Vol 48 (3) ◽  
pp. 386-388 ◽  
Author(s):  
M. Yaguchi ◽  
M. B. Perry
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Citrate Buffer ◽  
Neutral Amino Acids ◽  
Amino Acid Analyzer

An amino acid analyzer is used to separate seven 2-ammo-2-deoxy-D-hexoses (allosamine, altrosamine, glucosamine, mannosamine, gulosamine, galactosamine, and talosamine). A borate citrate buffer at pH 7.24 is used at 60 °C. Acidic and neutral amino acids, if present, are eluted before any of the hexosamines.

ION EXCHANGE CHROMATOGRAPHY OF THE FREE AMINO ACIDS IN THE PLASMA OF INFANTS UNDER 2,500 GM AT BIRTH

PEDIATRICS ◽  
1970 ◽  
Vol 45 (4) ◽  
pp. 606-613
Author(s):  
Johanne C. Dickinson ◽  
Herman Rosenblum ◽  
Paul B. Hamilton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Free Amino ◽  
High Sensitivity ◽  
Exchange Chromatography ◽  
Chromatographic Technique ◽  
The Individual ◽  
Amino Acid Concentrations

The free amino acids in the plasma of 46 infants who were under 2,500 gm at birth were determined by an ion exchange chromatographic technique of high sensitivity and resolution. Ninety-two plasma samples were collected from the 46 infants on different days after birth, and the data for 23 amino acids plus taurine and ethanolamine were summarized and compared with newborn, full-term and adult levels. In 16 cases tyrosine levels were high; these values are listed separately. With respect to the remaining amino acids, many showed marked changes during the first few postnatal days; but, by the end of the first week, stable patterns had developed. The decrease or increase of the individual amino acid concentrations in these infants compared to infants with birth weights over 2,500 gm and to the adult was not great and seemed to be characteristic for each amino acid. Attention was drawn to the technical details of preparing and analyzing physiological fluids which would minimize the changes in amino acid concentrations resulting from improper handling.

scholarly journals o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1969 ◽  
Vol 112 (5) ◽  
pp. 609-616 ◽  
Author(s):  
W. S. Pierpoint
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plant Extracts ◽  
Thiol Group ◽  
Amino Group ◽  
Amino Groups ◽  
Terminal Amino Acid ◽  
Secondary Reactions ◽  
Terminal Amino ◽  
Group 5

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their α-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The ∈-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their α-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from β-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.

scholarly journals Impact of C-terminal amino acid composition on protein expression in bacteria

2019 ◽  
Author(s):  
Marc Weber ◽  
Raul Burgos ◽  
Eva Yus ◽  
Jae-Seong Yang ◽  
Maria Lluch-Senar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Expression ◽  
Translation Termination ◽  
Terminal Sequence ◽  
Bacterial Taxonomy ◽  
Terminal Amino Acid ◽  
Protein Levels ◽  
Terminal Amino ◽  
The Impact

AbstractThe C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine) while hydrophobic amino acids and threonine are lower. In highly abundant proteins, the C-terminal residue is more conserved. We then studied the impact of C-terminal composition on protein levels in a library of M. pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to 4-fold. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels and is under selective pressure in highly expressed proteins.

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