Methionine radiolabelling

The suppression and recovery of protein synthesis from DTT treatment (without cycloheximide treatment) can be monitored via metabolic pulse radiolabeling of cell cultures using [35S]-methionine and subsequent determination of radiolabeled protein content either by SDS-PAGE/ phosphor-imager analysis or liquid scintillation of tricholoroacetic acid insoluble material (Stephens et al., 2005). 

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. 

Figure 6. Partial purification of Inhibitors I and II mRNA. Fractions containing Inhibitors I and II mRNA determined by in vitro translation analyses were recovered from an initial 15-30% linear sucrose gradient, precipitated by cold ethanol, and applied to a 10-25% linear sucrose gradient. The sample was centrifuged for 36 h at 25,000 rpm. Fractions of the gradient were collected and subjected to in vitro translation analyses. The upper graph represents total methionine incorporation assayed with 1 jiL of the translation mixture as described (ll). The bottom figure quantitates the radiolabel incorporated specifically into Inhibitor I (solid bars) and Inhibitor II (open bars).

In relation to their biosynthesis, it was proposed that A-B units of Et s are most likely formed by condensation of two Dopa-derived building blocks and that the tetrahydroisoquinoline ring in unit B is closed by condensation (Pictet-Spengler) with a serine(or glycine)-derived aldehyde, as in the case of the related saframycins. S-Adenosylmethinonine is the likely source of the methyl groups. A plausible route for the formation of unit C was proposed later [75]. This was partially demonstrated by incorporation of radiolabeled tyrosine and cysteine by Kerr and Miranda [80] and by incorporation of labeled methionine, glycine and tryptophan by Morales and Rinehart [81]. 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

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-... 

The biosynthesis of pilocarpine in Pilocarpus pennatifolius was studied by the administration of radiolabeled precursors. Radioactive sodium acetate, histidine, histidinol, methionine, and threonine were administered by the cut-stem method. Histidine, methionine, and threonine were administered together by a wick inserted through the stem of an intact plant. Sodium acetate and histidine were fed to root cuttings by suspending the roots in aqueous solutions of the precursors. After 64 - 7 5 h, the roots were harvested and total alkaloid extracts made. These extracts were then fed to stem cuttings. 

The biosyntheses of STA and REB were studied by feeding radiolabeled precursors to Lentzea albida (formerly Streptomyces staurosporeus) [9-13] and Lechevalieria aero-colonigmes (formerly Saccharothix aerocolonigenes) [14,15], respectively. The results established that the indolocarbazole core was derived from two units of tryptophan (with the carbon skeleton incorporated intact), while the sugar moiety was derived from glucose and methionine (for methylations). 

Preparation for Bioassav. The standard in vitro assay for allatostatins (1) is a two hour organ culture of corpora allata during which the inhibition of release of radiolabeled JH III from [ H-methyl] methionine is measured accordingly, the test sample must be free from unphysiological contaminants before incorporation into the modified tissue culture medium 199 (1). Our preparative-scale procedures all involved volatile modifiers (hydrochloric acid, formic acid, ammonium formate, TFA) which could be removed along with the solvents (ethanol, water, acetonitrile) this analytical workup of aliquots for bioassay was the only occasion when we used vacuum evaporation. 

Radiolabelling is often performed by oxidation of iodide by different agents for iodination of tyrosine and histidine residues in peptides. However, unfavourable oxidation reactions can occur simultaneously, e.g., cysteine and methionine can be oxidized to their corresponding sulphone or sulphonic acid, so that neutral amino acids are converted to highly acidic groups, thus changing the electrostatic distribution within the structme of the... 

Oorsprong, M.B.M. Kuiper, H.A. Toxicology In Vitro, in press). This could be demonstrated by using buthionine-S-sulfoximine, a specific inhibitor of GSH-synthesis, but so by using cultures containing S-labeled GSH, achieved by preincubation with S-methionine. In the latter case, there was a dose-related, increased loss of radiolabeled GSH, again in the absence of an effect on absolute GSH-levels (Figure 2). This effect may offer an explanation for the increased GSH-levels observed in livers from salmons and chars treated with furazolidone (72). 

Figure 2. Effect of furazolidone on intracellular GSH-levels. Prior to the exposure, cells were incubated with S-methionine, resulting in the labelling of GSH. Following exposure, amounts of both total (I I) and radiolabeled GSH were determined.

A similar compound, 5,8,11,14-eicosatetraynoic acid, is also an inactivator of 15-lipoxygenase (Kuhn et al., 1984). In contrast to the results obtained with 14,15-DHA, no radiolabeling of the enzyme by the [/nef/i> /- C]methyl ester of the tetradehydro analog was observed. Instead, amino acid analysis of the inactive enzyme revealed the presence of 1 mol of methionine sulfoxide, suggesting that loss of activity resulted from oxidation of the active site rather than covalent modification. 

---