Amino acid mixture analysis by mass spectrometry in the form of their dimethylaminomethylene methyl esters

Horman, Ian; Hesford, F.

doi:10.1002/bms.1200010206

Cited by 24 publications

(12 citation statements)

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“…Isotopic enrichment in glutamate, aspartate, and alanine was monitored using ratios of ions at m/z 433/432, 419/418, and 261/260, respectively. Enrichment in [2-15 N]glutamine was determined by monitoring m/z 259/258, and [5- citrulline was determined using the N-dimethylaminomethylene methyl ester derivative (38). Briefly, an aliquot of a tissue extract was purified via an AG-50 (H ϩ ; 100 -200 mesh; 0.5 ϫ 2.5 cm) column, dried, and then converted into N-dimethylaminomethylene methyl ester derivatives (38).…”

Section: Gc-ms Methodology and Determination Of 15 N-labeled Metabolimentioning

confidence: 99%

“…Enrichment in [2-15 N]glutamine was determined by monitoring m/z 259/258, and [5- citrulline was determined using the N-dimethylaminomethylene methyl ester derivative (38). Briefly, an aliquot of a tissue extract was purified via an AG-50 (H ϩ ; 100 -200 mesh; 0.5 ϫ 2.5 cm) column, dried, and then converted into N-dimethylaminomethylene methyl ester derivatives (38). 15 N enrichment in N-acetylglutamate in liver or mitochondrial extracts were determined using GC-MS and an isotope dilution approach (12).…”

Section: Gc-ms Methodology and Determination Of 15 N-labeled Metabolimentioning

confidence: 99%

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The Role of Mitochondrially Bound Arginase in the Regulation of Urea Synthesis

Nissim

Luhovyy

Horyn

et al. 2005

Journal of Biological Chemistry

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Section: Gc-ms Methodology and Determination Of 15 N-labeled Metabolimentioning

confidence: 99%

Section: Gc-ms Methodology and Determination Of 15 N-labeled Metabolimentioning

confidence: 99%

The Role of Mitochondrially Bound Arginase in the Regulation of Urea Synthesis

Nissim

Luhovyy

Horyn

et al. 2005

Journal of Biological Chemistry

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“…The residue recovered from the Dowex 50 column was transferred to a 1-ml reaction vial with ethanol. The ethanol was removed under a stream of nitrogen and the dimethylaminomethylene ethyl ester derivative of the amino acid was prepared by heating the residue in a mixture of 100 Ml of dimethylformamide diethylacetal, 60 Ml of acetonitrile, and 30 Ml of ethanol at 1000 for 15 min (21,22).…”

Section: Methodsmentioning

confidence: 99%

Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

Cohen¹,

Gruenke²,

Craig³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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a-l-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the a-l-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the a-l-antitrypsinsin complex can be split at high pH, releasing a component of a-l-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the a-l-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 180H. When the new carboxy-terminallysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the a-l-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on a-l-antitrypsin bind covalently to each enzyme.Serine proteases cleave proteins by binding to the amino acid for which they are specific and forming a series of intermediates including a tetrahedral adduct, an acyl ester, and, finally, two fragments of the protein and the free enzyme (1-4).On the basis of many structural studies of protein inhibitors of proteolytic enzymes, Bode and his colleagues (5) concluded that the complexes that have been studied by x-ray crystallography are intermediates in peptide hydrolysis (6, 7). Soybean trypsin inhibitor is much larger than pancreatic trypsin inhibitor and folds differently. However, analysis of the complex between porcine trypsin and soybean trypsin inhibitor showed a closely similar conformation of the contact segment. A tetrahedral adduct formed in both cases. Studies of the interaction of trypsin with a-l-antitrypsin, a human serum protein, have suggested that the catalytic site of the enzyme interacts with the active site of this protease inhibitor (8). Moroi and Yamasaki (9) found that the enzyme-inhibitor complex had a lower molecular weight than the constituent reactants, that a-iantitrypsin released from the complex by hydrazine had lost a peptide, and that the amino-terminal residues of the complex were identical to those of trypsin and a-l-antitrypsin, indicating that a-l-antitrypsin had lost a peptide from its carboxyl end. The findings suggest that a-1-antitrypsin is bound to trypsin through an acyl ester bond. Characteristics of this type of bond have been studied with low molecular weight acyl esters.Alkaline hydrolysis of carboxyl esters in aqueous solution is known to follow second-order kinetics and to involve acyloxygen fission (11). Alkaline hydrolysis of n-amyl acetate in solvent water enriched in 180 showed that the oxygen from the medium does not appear in the alcohol produced but resides in the acid (12) and, therefore, that the mec...

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“…(N, N)-Dimethylformamide dimethyl acetal (DMFDMA) derivatization (modified from Horman and Hesford, 1974;Thenot and Horning, 1972). The dried hydrolysate was dissolved in 100 +L methanol.…”

Section: Preparation Of Amino Acid Derivativesmentioning

confidence: 99%