Tritiated amino acid

JM Manning. Determination of D- and L-amino acid residues in peptides. Use of tritiated hydrochloric acid to correct for racemization during acid hydrolysis. J Am Chem Soc 92, 7449, 1970. 

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. 

It is evident that the studies with tritiated proline do not permit definite conclusions about the mechanism of hydroxylation further information is needed about the labeling of the proline used and possible isotope effects. It is probable that there is some randomization of label during tritiation of 3,4-dehydroproline (40), and apparently also some racemization. Thus, we have observed that the tritiated proline preparations contain 10 to 20% D-proline and that oxidation by D-amino acid oxidase releases significant amounts of tritiated water. 

Autoradiography is a technique for locating radioactive compounds within cells it can be conducted with light or electron microscopy. Living cells are first exposed to the radioactive precursor of some intracellular component. The labeled precursor is a compound with one or more hydrogen ( H) atoms replaced by the radioisotope tritium (3H) e.g., [3H]thymidine is a labeled precursor of DNA, and [3H]uridine is a labeled precursor of RNA (Chap. 7). Various tritiated amino acids are also available. The labeled precursors enter the cells and are incorporated into the appropriate macromolecules. The cells are then fixed, and the samples are embedded in a resin or wax and then sectioned into thin slices. 

The working substance of the ITEP-group /9 source was a doubly tritiated crystalline amino acid, DL-valine (Mallikarjunan and Rao, 1969), which is a molecular crystal. As a result of decay, during the time 10 18 sec the valine molecule transforms into the corresponding helium-containing ion (RHe)+. Since the translational symmetry of the crystal is disturbed, no exciton states are produced, and only molecular states of the complex (RHe)+ are excited. Since in molecular crystals the intermolecular interactions are considerably weaker than the intramolecular ones, one can neglect the influence of the valine crystalline surrounding on the /S-decay process and consider only an isolated valine molecule. 

Scheme VI. Stereochemical possibilities in the reduction of an L-amino acid-transaminase equilibrium complex with tritiated NaBH4.

Tonoiilament Assembly. The available information suggests that keratin is sequentially assembled around primary fibers that originate within the attachment plates of desmosomes (27). The dense cores of 50-A filaments (stained with uranyl acetate) represent such fibers (64, 84). These cores and their surrounding fibrous protein probably contain about 50% a helix (71). They contain even less sulfur (32, 64) than the high methionine (1.4 residues/100 amino acid residues), low cystine (1.1 residues/100 amino acid residues) fraction obtained by Baden after partial enzymatic hydrolysis (82). Studies with tritiated amino acids suggest that basal cells preferentially incorporate methionine, leucine, and phenylalanine within their elements (73, 74). In Figure 13A the primary rope is identified with the 35-A diameter of the smallest filaments that have been isolated (32). 

Inactivation of alcohol dehydrogenase from yeast with 14C-labeled [3-(3-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation showed the presence of 1-carboxymethyl histidine66. After inactivation of the enzyme with labeled [3-(4-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation, S-carboxymethyl cysteine was identified in the protein. In the case of glyceraldehyde-3-phosphate dehydrogenase, treatment with either coenzyme analogue leads to the modification of the cysteine residue. Treatment with [14C]nicotinamide-5-bromo-4-methylimidazole dinucleotide did not reveal any modified amino-acid-residues. The labeled nicotinamide residue split off during the recovery of the inactivated enzyme. Attempts to synthesize an inactivator labeled with a 14C-acetyl residue did not give satisfactory yields. If the enzyme-coenzyme derivative was treated with tritiated sodium boron hydride, tritium could be introduced (Fig. 22). Studies with... 

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

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