14C-Labelled substrate

Experiments with 14C-labelled substrates also demonstrated conclusively the intramolecularity of the rearrangement. The generally accepted scheme involves the formation of the dienone LXXXV which can lose a hydrogen atom if R = H, to form the ortho product or if R H further rearrangement to LXXXVI occurs with subsequent formation of the para product, viz. 

The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The label is then chased through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway. 

Durall D. M., Todd A. W., and Trappe J. M. (1994) Decomposition of 14C-labelled substrates by ectomycor-rhizal fungi in association with Douglas fir. New Phytol 127(4), 725-729. 

The design of the uptake experiments themselves and the analytical determinations are straightforward specific analysis may be carried out for the compounds being examined (together with their metabolites) or advantage may be taken of, for example, 14C-labeled substrates. A number of important limitations in the numerical significance of the values obtained in laboratory studies have been pointed out (Oliver and Niimi 1985) and these are worth emphasizing ... 

All these observations emphasize that tests for biodegradability carried out at high substrate concentrations may not adequately predict the rates of degradation occurring in natural ecosystems where only low concentrations of xenobiotics are encountered (Alexander 1985). This phenomenon is therefore of enormous environmental importance since it would imply the possibility of extreme persistence of low concentrations in natural ecosystems. The further exploration of this phenomenon is probably only limited in practice by the access to analytical methods for measuring sufficiently accurately substrate concentrations at the level of ng/1 or lower. Most studies that have been carried out have therefore used 14C-labeled substrates which necessarily limits the range of compounds accessible and restricts the elucidation of metabolic pathways in which only biotransformation or partial mineralization has occurred. 

Microcosms are laboratory systems generally consisting of tanks such as fish aquaria containing natural sediment and water, or soil. In those which have been most extensively evaluated for aquatic systems, continuous-flow systems are used. In all of them, continuous measurement of 14C02 evolved from 14C-labeled substrates may be incorporated, and recovery of both volatile and nonvolatile metabolites is possible so that a material balance may be constructed (Huckins et al. 1984). It should be pointed out that the term microcosm has also been used to cover much smaller scale experiments that have been carried out in flasks under anaerobic conditions (Edwards et al. 1992), and to systems for evaluating the effect of toxicants on biota (Section 7.4.2). Some examples are given to illustrate different facets of the application of microcosms to study various aspects of biodegradation. 

The fate of 2,3,7,8-tetrachlorobenzo[l,4]dioxin (TCDD) was examined in freshwater ponds using 14C-labeled substrate over a period of up to 2 years. Equilibrium concentrations in pondweeds (Elodea nuttali and Cerotophyllon demersum), in fathead minnows (Pime-phales promelas) and in sediment were attained after 1, 2, and 6 months, respectively. After 1 year, most of the remaining TCDD was found in the pondweed, and after 2 years, through death of the plants, almost all the TCDD was found in the sediment phase that included plant detritus. Unidentified metabolites were confined to the aqueous phase and the plants (Tsushimoto et al. 1982). 

C]labeled substrates were synthesized according to the trifluoroacetic anhydride method described by Kanada and Wells (1981). 

In vitro and ex vivo studies have shown that FATPs transport LCFAs and very long-chain fatty acids (VLCFAs) but no medium-chain fatty acids, fatty acid esters, or lipid-soluble vitamins [4]. LCFA transport is inhibited by prior protease treatment. Synthetic substrates for FATPs include 14C-labeled fatty acids and the fluorescently labeled fatty acid analogue C1 -BODEP Y-Cl 2. Using the latter substrate, differences in fatty acid uptake kinetics between FATP expressing 3T3 LI adipocytes and 3T3 LI fibroblasts, which are devoid of FATPs, can be readily appreciated (Fig. 2). 

Another method is based on the same principle,112 in which the [14C]labelled methyl ester of D-galacturonan is prepared by esterification of pectic acid with [,4C]diazomethane. In the course of the enzymic de-esterification, aliquots are removed, and the unreacted substrate is precipitated with acidified ethanol or 1-propanol. After centrifugation, the labelled methanol in the supernatant liquor is determined in a liquid scintillation counter. An advantage of this method lies in the possibility of using, as substrates, short-chain oligo-D-galactosiduronates partially esterified with [14C]methanol. These substrates, beginning with the trisaccharide, are not soluble in 1 4 80% phenol-diethyl ether, which is used for the extraction of enzymically released, labelled methanol. 

The doubly nitrogen-15 labelled substrate required for determining the nitrogen isotope effect for this reaction was obtained by the reactions shown in Scheme 2340. The series of reactions used in the synthesis of the [4,4 -13C2]-4,4 -dichlorohyrazobenzene is shown in Scheme 2440, and the preparation of the [2-14C]- and the [4-14C]-4,4 -dichlorohyrazobenzene are described in Schemes 25 and 26. 

In brief, the rats are anesthetized, followed by an injection of 0.2 mL of the test solution into the common carotid artery. The injection solution consists of a HEPES buffered Ringer s solution (containing 141 mM NaCl, 4 mM KC1, 2.8 mM CaCl2, and 10 mM HEPES, pH 7.4) which contains both the test substrate (e.g., a [3H]-labeled compound, about 10 /xCi) and a reference compound, which is highly extracted by the tissue (e.g., 0.1 /xCi [14C]n-butanol) in the presence or absence of transport inhibitors. If a [14C]-labeled compound is used as a test substrate, [3H]H20 can be selected as a reference compound. Rats are decapitated at 15 s after injection and the retina is removed. The retina is dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity is measured by liquid scintillation spectrometry. The RUI value, an index of the retinal distribution characteristics of the [3H] test substrate, is estimated using the following relationship ... 

Figure 4. Specific activity of cutinase for the T-8 parental strain and PNB-1 mutant strain after growth on medium containing 200 mg cutin. Enzyme activity was assayed with the artificial substrate PNB (Panel A) and with the natural substrate, [14C]-labelled cutin (Panel B). (Reproduced with permission from Ref. 13. 1986, American Society for Microbiology.)...

Separation of radioactively labeled substrate and product on diethylaminoethyl (DEAE) cellulose 14C-galactose is eluted by H20, 14C-galactose-l-phosphate is retained 14C-galactose-l-phosphate is subsequently eluted by 100 mM HC1. Determination of eluted radioactivity in a liquid scintillation counter (/1-counter) [5,... 

Separation of the radioactively labeled product from the labeled substrate on a DEAE cellulose column with 20 mM HC1,14C-galactose-l-phosphate is eluted and UDP-14C-galactose is retained. The latter is eluted with 100 mM HC1. Radioactivity is determined with the aid of a scintillation counter (/ -counter) [unpublished]. 

Treatment with sodium borohydride of the enzyme-substrate complex of aldolase A and dihydroxyacetone phosphate leads to formation of a covalent linkage between the protein and substrate. This and other evidence suggested a Schiff base intermediate (Eq. 13-36). When 14C-containing substrate was used, the borohydride reduction (Eq. 3-34) labeled a lysine side chain in the active site. The radioactive label was followed through the sequence determination and was found on Lys 229 in the chain of 363 amino acids.186/188 188b Tire enzyme is another (a / P)8-barrel protein and the side chain of Lys 229 projects into the interior of the barrel which opens at the C-terminal ends of the strands. The conjugate base form of another lysine,... 

This experiment has been extended by using the double-labeled substrate [14C]Leu-Tyr-[3H]Leu to show that simultaneous amino and acyl transfer could take place. Both [3H]Leu-[3H]Leu and [14C]Leu-[14C]Leu are formed.168 The 14C-labeled product, which predominates by a factor of 3 or 4, could come from the acyl transfer route, whereas the 3H-labeled product could arise from the [14C]Leu-Tyr-[3H]Leu-[3H]Leu produced from an aminoenzyme by mechanism 16.32, where E-[3H](NH-Leu) is [3H]Leu bound to E by the NH group of the amino acid. 

The reactions were carried out under standard conditions using the specifically tritiated substrate mixed with uniformly 14C labeled TDP-glucose. Samples were taken at different time intervals and the reaction products were isolated. 14C activity was used to measure the reaction coordinate. The reactions were permitted to proceed until isotopic equilibrium was achieved. 

Microorganisms or enzyme systems prepared from them have been used to produce organic acids labeled with 14C, either by direct synthesis or transformation of labeled substrates. Several species of microorganisms have been used to produce higher fatty acids by condensation. 

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