Methionine, isotopically labelled

Until recently, cell-free protein expression (also sometimes erroneously named in vitro protein expression) did not exhibit the productivity required for preparation of NMR samples, especially considering the high cost of using isotopically labeled starting material. Rather, it was exclusively used as an analytical tool that served to verify correct cloning or to study promotor sites. Because of the very low yields, detection of the expressed product usually required incorporation of a radioactive label (usually via 35S-methionine). 

A more extensive study of mobilities of 3H- and 14C-labeled amino acids again found that amino acids labeled with 14C at Cl or C2 are retained on the column, relative to the unlabeled forms.135 Lysine is an exception. Tritiation at C3 also increases the retention time, but tritiation at C2 of glycine or at C4, C5, or C6 of lysine decreases it, and large decreases are seen with methionine tritium-labeled in the methyl and with tyrosine tritium-labeled at C3, 5. The 14C IEs can be attributed to a decrease of acidity, but the IEs of distant 3H may be due to hydrophobic interactions with the resin. A remarkable result is that intramolecular isotopic isomers (isotopomers) can be distinguished on the basis of their chromatographic mobilities. 

Dissection of the chemical structure of jamaicamides A-C led to the speculation that these metabolites derive from a mixture of polyketides (nine acetate units), amino acids (t-Ala and p-Ala), and the S-methyl group of methionine. To map out the biosynthetic subunits of these molecules, isotopically labeled precursors were supplied to I. majuscula JHB, and the labeling patterns discerned by NMR spectroscopy (Figure 6.12). From these experiments, insights were gained into the biochemical transformations that produce the jamaicamides, especially the mechanism of formation of the vinyl chloride group [157]. 

The SI LAC approach has also been used to investigate metastatic prostate cancer development at the protein level (Everley et al., 2004). The fact that proteins showed altered concentration ratios by quantitative MS was confirmed by western blotting. In addition, proteomic approaches for quantitation of protein phosphorylation via SILAC combined with MS analysis have been described (Gruhler et al., 2005 Ibarrola et al., 2003, 2004). A recent study reports on identification as well as relative quantitation of in vivo mefhylation sites of proteins in HeLa cells by stable isotope labeling wifh C Hj-methionine (Ong et al., 2004). 

The relation of many of the simpler alkaloids to the aromatic amino acids is obvious. For example, germinating barley contains (241), besides tyrosine and tyramine, A -methyltyramine, JViV -dimethyltyramine (hordenine), and the trimethylammonium derivative (candicine). In this simple case the. AT-methylated derivatives are known to be derivable from isotopically labeled tyramine (538) and the methyl groups are known to arise from methionine by transmethylation (540, 586). Similarly AT-methyl derivatives of phenylethylamine, 3,4-dihydroxyphenylethylamine, and 3,4,5-trihy-droxyphenylethylamine are well known alkaloids (cf. review, 701). N-Methylated derivatives of tryptamine and hydroxytryptamine equally occur for example, eserine has an obvious relation to 5-hydroxy tryptamine. Methylated derivatives of metabolites of the aromatic amino acids also occur, for example, trigonelline (67), which is the betaine of nicotinic acid, and damascenine is probably similarly related to hydroxyanthranilic acid. 

DL-Selenomethionine was initially synthesized by Painter" via a sodium/liquid ammonia reduction of DL-selenohomocystine followed by an alkylation of the resulting sodium selenohomocysteinate with methyl iodide. Other syntheses have since been reported including synthetic pathways for the preparation of optically active material" " and isotopically labeled material "" . DL-Selenomethionine has a solubility in water at 30° and pH 7 of 0.108 M which is considerably less than that for L-methionine (0.386 M). After seven hours of hydrolysis under anaerobic conditions in 6 N HCl at 110 °C selenomethionine is completely decomposed (under the same conditions 96% of methionine remains). Chemically, selenomethionine appears to be more reactive than methionine . For example, with cyanogen bromide, selenomethionine completely reacts in 0.1 M HCl in fifteen minutes while methionine requires twenty-four hours for the same reaction. In both cases the end product is homoserine. Although not as marked, this difference in reactivity was also confirmed in the reaction with hydrogen peroxide . 

Shen, M. Guo, L. Wallace, A. Fitzner, J. Eisenman, J. Jacobson E. Johnson, R.S. Isolation and isotope labeling of cysteine- and methionine-containing tryptic peptides application to the study of cell surface proteolysis. Mol. Cell. Proteomics 2003, 2, 315-324. 

From feeding experiments with isotopically labelled 5-aminolevu-linic acid (18) and methionine (142) it became clear that the carbon skeleton of the naturally occurring highly saturated porphyrins are formed from uroporphyrinogen III (14) and that the additional methyl groups are transferred from 5-adenosyl methione (77, 72). 

Me Intyre CR, Scott FE, Simpson JT, Trimble LA, Vederas JC (1989) Application of stable isotope labelling methodology to the biosynthesis of the mycotoxin, terretonin, by Aspergillus terreus incorporation of C-labelled acetates and methionine, and C, 0-labelled 3,5-dimethylorsellinate and oxygen-18 gas. Tetrahedron 45 2307-2321 Me Lean SM, Mahler P, Nyburg SC, Sawyer JF, Webster CJ, Wong-NG W (1983) Structure of rhodocladonic acid. X-ray crystal structure analysis of its triacetate. Can J Chem 61 2055-2058... 

Many of radioactive isotopes are very useful for the following biochemical processes (Table 6.1). The radioactive label is introduced into macromolecules, especially proteins, either during biosynthesis, e.g., during translation in the presence of S-methionine, or enzymatically, e.g., by use of P-labeled ATP during protein phosphorylation by protein kinases, or chemically by modification of amino acid side chains. Examples for reagents used in chemical radiolabeling of proteins are given in Table 6.2. 

The biosynthesis of virginiamycin Ml in Streptomyces virginiae has been studied using both radiolabeled precursors (63) and stable isotope techniques (45, 63-65). Incorporation of [2-l4C]acetate, L-[methyl-3H]methionine, dl-3-14C]serine, L-[3,4-3H2]proline, and [2-14C]glycine established these compounds as the main precursors (63). The assumption that carbons 2, 26, 27, and 28 arose from valine was supported by the observation that no incorporation of appropriately labeled mevalonolactone was observed (65). On the basis of the radio-... 

Stable isotope methodology has been applied to the study of the biosynthesis of madumycin II (A2315A, 92) in Actinoplanes philippensis (60, 64). As with virginiamycin Ml (90), carbons 2, 26, 27, and 28 were found to be derived from valine, C-29 from methionine, C-3 to C-6 from acetate, N-7, C-8, and C-9 from glycine, carbons 10 to 17 and C-31 from acetate, and N-18, C-19, C-20, 0-21, C-32, and 0-34 from serine. The origin of the D-alanine residue, N-23, C-24, C-25, C-35, and 0-36, was of particular interest in this study. No incorporation of DL-[U-13C]serine was observed in the alanine portion of the molecule, eliminating the intermediacy of the a,(3-dehydro alanine unit 101 derivable from the acylserine precursor 100. This was corroborated by the observed incorporation into the molecule of intact doubly labeled L-[3-l3C,3,3,3-2H]alanine. dl-[1-l4C]Alanine was also efficiently incorporated. These results and those from de-... 

The specific activity of proteins assayed by direct immunochemical methods or those used as standards in radioimmunoassays profoundly affects the resolution attainable by these techniques. Therefore, the nature of the radioactive label to be used in such experiments must be considered carefully. Table 8-6 lists the isotopes available for this purpose along with the number of atoms of each isotope that must be incorporated to produce an arbitrary counting rate. As can be seen here, 557 atoms of H and 261,672 atoms of C must be incorporated into every molecule of protein to yield the same number of disintegrations per minute as only one I or 11 S molecules. S-methionine is often the isotope of choice for many direct immunochemical procedures since it is relatively inexpensive to prepare at high specific activity. On the other hand, the relative ease with which radioactive iodine may be incorporated into a purified antigen makes it the isotope of choice for radioimmunoassay methods. Of the two iodine isotopes available, is most often used because of its longer half-life. This is an important consideration since it usually takes more than 1 week to prepare and test a labeled antigen prior to its experimental use. 

Recombinant human leptin was recently cloned (8) and expressed in E. coli, and demonstrated to effectively regulate adiposity in mice through modulation of appetite and metabolism (9, 10). The molecule contains four methionine residues at positions 1, 54, 68, and 136. In this paper, we report the separation and characterization of three norleucine-incorporated recombinant human leptins which were uniformly labeled with 15n isotope or double labeled with and isotopes. The extent of incorporation at each methionine residue can be determined by reverse-phase HPLC and amino acid analysis methods. The norleucine incorporation was observed preferentially occurring at the internal Met residues. 

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