Labeling transamination

Experiments with (2S,3R)- and (2/, 35 )-[3- H]phenylalanines gave tritium retentions of 44% and 24%, respectively (Vederas and Tamm, 1976). Simultaneous incorporation of equal amounts of both enantiomers led to the expected 34% retention of hydrogen label. Transamination occurs stereo-specifically at position 2 of the amino acid therefore, the participation of at least two enzymes with different stereochemical requirements at the 3 position is reasonable. Two biosynthetic pathways are consistent with the data available (see Fig. 13). Path A in Fig. 13 depicts (2 S )-phenylalanine as the actual precursor which is in rapid equilibrium with its enantiomer in path B phenylpyruvic acid (49), derived directly from shikimic acid, is the primary precursor. Considerable suppression of the incorporation of D-amino acid by phenylpyruvic acid (49) indicated that the naturally abundant L-enantiomer is the actual primary precursor, thus demonstrating that path A (Fig. 13) is probably the main biosynthetic route. Both enantiomers are in rapid equilibrium with phenylpyruvic acid (49) via the action of aminotransferases or amino acid oxidases. The stereochemistry of hydrogen loss... 

Protocol for Labeling Nucleic Acids by Bisulfite-Catalyzed Transamination... 

The biological importance of transamination was confirmed using 15N-labeling experiments (Tannenbaum and Shemin, 1950). 15N-leucine incubated with pig heart muscle gave highly labelled 15N-glutamate, evidence that leucine could be transaminated. Isotope experiments were then extended to the whole range of amino acids. 

Back in 1937, Braunstein and Kritzman and, independently, Herbst, had proposed transamination might proceed via a Schiff s base formation. The essential lability of the H atom on the a-C atom was shown with deuterium labelling (1942). a-2H alanine released 2H into the medium during transamination. The label did not appear in glutamate, the end-product. 

Answer In maize, C02 is fixed by the C4 pathway elucidated by Hatch and Slack. Phospho-enolpyruvate is rapidly carboxylated to oxaloacetate, some of which undergoes transamination to aspartate but most of which is reduced to malate in the mesophyll cells. Only after subsequent decarboxylation of labeled malate does 14C02 enter the Calvin cycle for conversion to glucose. The rate of entry into the cycle is limited by the rate of the rubisco-catalyzed reaction. 

One third of the label from A1-[6-14C]piperideine that was found in the matrine (32) was located elsewhere than at C-2, C-10, and C-15. A plausible explanation for the partial randomization that was observed is that reversible transamination converted the AMfi- Clpiperideine [as (31)], via its ring-opened form (30), into cadaverine (25). Subsequent re-conversion into (31) gave material that was labelled at C-6 and also at C-2.35... 

The production of aldehydes and alcohols from the hydrophobic amino acids has been studied primarily in yeast (147,148,149,154) since the fusel oils produced are significant secondary products of alcoholic fermentation (149). Also important are the studies of Morgan and coworkers on the malty flavor defect in milk which is produced by Streptococcus lactis var. maltigenes (155-161). This flavor is principally caused by 3-methylbutanal (155) which is produced by transamination of leucine to a-ketoisocaproate and decarboxylation (157). Labeling experiments showed that this compound is produced from leucine by tomato extract (9, 162, 163). The corresponding 3-methylbutanol and 3-methylbutyl acetate are produced similarly in banana (145, 163). Similar transformations of valine and phenylalanine are also carried out by banana slices (145). 

Scheck RA, Francis MB. Regioselective labeling of antibodies through N-terminal transamination. ACA Chem. Biol. 2007 2 247-251. 

Transamination simple introduction of reactive amino groups in cytosine on ss strands many primary or secondary labels may be reacted with this nucleophile useful for universal probes... 

Acridinium-NHS (AT-hydroxysuccinimide) ester labeling reagent is synthesized as described by Weeks et al. (1983) and probes containing an allylamine linker arm (Fig. 7.2.1) are prepared either as described for nucleotides and for enzymatically labeled probes (Section 7.6.2.2), by transamination (Section 7.8.1) or during oligomer synthesis (Section 6.4). The acridinium-NHS ester is then reacted with the linker arm by standard methods (Section 7.4.1). 

Labeling of nucleic acids via JV-hydroxysuccinimide (NHS) esters of fluorescein (Research Organics) or Eu -chelates is straightforward. Nucleic acid should then possess reactive amino groups. These can be introduced by transamination (Section 7.8.1) or by using amino-hexyl-dNTP or AH-NTP as precursors during enzymatic probe synthesis (Section 7.6.2.2 Folsom et al., 1989). After AH-NTP incorporation in RNA (or AH-dNTP in DNA and heat-denaturation), the nucleic acid is diluted to 1 p,g in 25 p,l of HjO and added to 25 xl of freshly prepared 0.4 M sodium bicarbonate (pH about 8.2) and then to 10 pel of ester in DMF (1.7 mg/ml of ester final concentration). After incubation for 2 h, the reaction is stopped by adding 50 g.1 of 1... 

In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

An interesting approach to selectively label regions outside the probe is to tall ds molecules (e.g., inserts from plasmids or PCR-pro-duced duplices) with C-homopolymers and to transaminate these tails without heat-denaturation of the duplex. Subsequent restriction of the duplex and purification of the fragments yields strand-specific probes. 

Although glutamic acid or aspartic acid is added to the artificial diet containing [ N]Ala, in order to avoid the transamination from Ala to these amino acids, the [ N]Ala labeling of the sample was still low [18]. Thus, another isotope labeling method is required. 

Figure 12.7-13. Production of lsN-labeled amino acids by transamination.

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