3H-lysine

Label cellular proteins metabolically by incubating cultures for 4-24 h with 35S-methionine, 3H-lysine, or l4C-amino acids in medium deficient in the relevant amino acid. 

Casein-Bound 3H Lysine in Untreated, Early, and Advanced Maillard Casein (41). Goat sodium caseinate biologically labelled with L-4,5-3H-lysine was treated with glucose under conditions inducing early and advanced Maillard reactions. The two preparations contained about the same reactive lysine (see Table III) the early Maillard 3H-casein contained only c-deoxyfructosyllysine as Maillard products and the advanced Maillard 3H-casein contained some c-deoxy-fructosyllysine and unidentified derivatives of lysine and glucose as brown pigments. 

Untreated, early Maillard and advanced Maillard (3H-lysine)-casein were given to groups of three rats each in a single meal and the radioactive urinary and fecal excretions were measured for 74 h. After this period, the animals were killed and the radioactivity of different organs was determined. 

For the untreated (3H-lysine) casein, the radioactive urinary and fecal excretions were low, about 10% and 4%, respectively, after 74 h. The unexcreted radiactivity was retained in the organism and incorporated into the proteins. The radioactivity measured in the liver, muscle, and kidneys (expressed as percentage of the ingested dose per gram of tissue) can be considered as a function of the quantity of lysine incorporated into the protein of these tissues and proportional to the biological availability of 3H-lysine in the casein samples. The value of radioactivity measured in the liver, muscle, and kidneys can be considered as being given by a protein whose lysine is 100% available (see Table III, Column 5). 

For the early Maillard (3H-lysine) casein, the urinary excretion was higher than for the untreated casein. This value represents the quantity of protein-bound c-deoxyfructosyllysine absorbed by the intestine. It is the same value as that determined in the previous experiment... 

Time was 74 h after a meal containing 3H-lysine casein, untreated, and heated under conditions for the early and advanced Maillard reaction (41 ). 

For the advanced Maillard (3H-lysine) casein, the urinary excretion was low and the fecal excretion high, suggesting that lysine engaged in advanced Maillard products is absorbed at a very low extent by the intestine and that the intestinal microflora does not metabolize these products. The radioactivity measured in the liver and in the muscle indicates that the biological availability of lysine in the advanced Maillard (3H-lysine)-casein is lower than that determined by the chemical analysis (reactive lysine). About 50% of reactive lysine should be not biologically available (see Table III, Columns 4 and 5). The level... 

Percentage of the value obtained by the untreated (3H-lysine) casein. 

Metabolic Transit of Free Radioactive e-(y-Glutamyl) lysine. The first metabolic study of this isopeptide was made by Waibel and Carpenter (81) who showed that this molecule was present in the blood plasma of chicks and rats receiving it in their diet. Using c-(y-glutamyl)-[4,5-3H] -lysine, Raczynski et al. (82) confirmed that the isopeptide passed unchanged across the intestinal wall into the serosal fluid in everted sacs and found that the kidneys were very active in hydrolyzing this isopeptide. These authors also found small hydrolytic activities in the intestinal mucosa and the liver. 

Figure 6. Urinary (top) and fecal (bottom) excretion of the radioactivity after oral administration in rats of (3H-lysine) casein untreated ( ) and treated with caffeic acid at pH 7 plus tyrosinase (A) and pH 10 ( ). Each curve corresponds to one rat each product was tested on three rats.

We are grateful to D. Salter of the National Institute for Research in Dairying, Shinfield, Reading, England for the labelling of 3H-lysine casein and R. Jost for its purification. Many thanks to I. Bracco for the whole-body autoradiographies and R. F. Hurrell for his advice and the correction of the manuscript. 

It has also been difficult to study the enzyme because native substrates and inhibitors of the enzyme are extracted with it into urea and thus must first be dissociated and removed before an estimation of true activity can be obtained (44,45). Further, there are no well-characterized substrates available for the routine assay of lysyl oxidase. The standard assay is a procedure described by Pinnell and Martin (31). The substrate is prepared from embryonic chick aortas after culture in vitro in the presence of 4,5- or 6-3H-lysine and inhibitors of endogenous lysyl oxidase (cf. Figure 3). 

Figure 3. Lysyl oxidase. The enzyme, lysyl oxidase, appears to seek out lysyl residues in alanyl- and lysyl-rich regions in the pro fibrillar forms of elastin. The presence of an aromatic amino acid residue adjacent to lysine appears to block its oxidation. The product of oxidation is peptidyl a-aminoadipic-S-semialdehyde. Assays for the enzyme against elastin involve first the preparation of an elastin-rich pellet containing 3H-lysyl residues labeled in the 6 or 4,5 position. This is usually accomplished by incubating embryonic chick aortas in medium containing 3H-lysine plus f3-aminopropionitrile (BAPN) to inhibit endogenous lysyl oxidase activity. BAPN is then removed leaving behind an elastin-rich residue in which the profibrillar forms of elastin labelled with 3H-lysine are only partially crosslinked. When lysyl oxidase preparations are added to this residue the release of tritium represents the assay for activity. It has also been demonstrated that tropoelastin, when incubated with lysyl oxidase, forms a-aminoadipic-S-semialdehyde and eventually crosslinks as shown in Figure 4.

H-Lysine accumulation into brain and plasma proteins in male rats treated with morphine, naloxone, or naloxone plus... 

The effect of morphine on the metabolic transport of 3H-lysine and incorporation into protein... 

To further exploit the potential usefiilness of this new family of clusters, monoadduct 54 was saponified into 55 (0.05 M NaOH, quant) and condensed to L-lysine methyl ester using 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ) to give extended dimer 56 in 50 % yield together with monoadduct in 15 % yield [75]. Additionally, tert-butyl thioethers 52 could be transformed into thiols by a two step process involving 2-nitrobenzenesulfenyl chloride (2-N02-PhSCl, HOAc, r.t, 3h, 84%) followed by disulfide reduction with 2-mercaptoethanol (60%). Curiously, attempts to directly obtain these thiolated telomers by reaction with thioacetic acid f ed. These telomers were slightly better ligands then lactose in inhibition of binding of peanut lectin to a polymeric lactoside [76]. 

Peptide aldehydes are fairly reactive and usually exist in solution partially as hydrates Xaa[CH(OH)2] (e.g., 3), which are identified using 13C NMR spectroscopy. 3 3" With amino acids containing nucleophilic side chains, cyclized forms predominate (Scheme 2). Argininal analogues such as Z-Leu-Phe-Arg-H have been shown to exist primarily as the carbinol amine 4 with no spectroscopic evidence for an iminium structure. Lysinal derivatives such as Z-Leu-Phe-Lys-H exist both as a cyclic carbinol amine 5 and as an iminium ion 6 as shown by 3H and 13C NMR spectra. The C-terminal ornithinal analogue Z-Leu-Phe-Orn-H exists primarily as the carbinol amine form 7 and the cyclic iminium form 8 as characterized by the 13C NMR spectra. 3,21 In general, the existence of carbinol and iminium cyclized forms does not preclude the existence of the free or hydrated aldehydes under aqueous conditions. 

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. 

The chirality of a precursor product relationship was determined by the use of doubly labeled lysine, in which one enantiomer was labeled only with tritium and the other with tritium and 14C (55). Comparison of the 3H/14C ratios of substrate and products demonstrated that decodine and decinine were derived from L-lysine, whereas pipecolic acid (186) was derived from D-lysine. Thus, pipecolic acid does not serve as a precursor of Lythraceae alkaloids (57). 

If less strong emitters than 125I are needed, labelling with 3H can be used. Various tritiated reagents are available, such as [3H] substituted N-hydroxysuccinimi-doesters (e.g., N-sucdnimidyl 2,3-[3H]propionate) that react with primary amino groups, either the N-terminus or e-amino groups of lysine. 

As an alternative strategy, lysine residues can be modified through reductive alkylation. Fig. 2e. This method is most frequently carried out by exposing the protein to aldehydes in the presence of hydride-containing agents that reduce the transiently formed imines. NaB(CN)H3 and NaB(OAc)3H are commonly used for this purpose. As an alternative, transfer hydrogenation can be carried out in the presence of an Ir(III)[Cp ]2(bipyridyl) catalyst, which allows imine reduction to occur under mild conditions using buffered formate as the hydride source (14). 

Further work56 has now disproved this pathway, at least for sedamine. When [2-3H,6-14C]lysine was administered to Sedum acre the alkaloid incorporated both labels with an unchanged isotopic ratio. Thus, the hydrogen at C(2) of lysine is not lost in the biosynthesis as would be required for pathway a. An alternative sequence, path b (Scheme 16) satisfactorily accommodates the fresh evidence. Decarboxylation at C-2 now precedes rather than follows the oxidation step. To avoid the generation of a symmetrical diamine, the amino-group at C(6) is differentiated by methylation in the first step. 

It has been established that nicotinic acid is the precursor of the pyridine ring in anabasine 140) and that carbons 2 and 3 are derived from the methylene carbons of succinate 127). It has also been shown that glycerol serves as a precursor for C-4, C-5, and C-6 119). Lysine serves as the precursor of the piperidine ring of anabasine 140, 141), C-2 of lysine becoming C-2 of anabasine. Tracer experiments with lysine-2- labeled with on the a- or e-nitrogen indicate that the piperidine nitrogen is derived from the e-amino group 142). The mechanism by which nicotinic acid is incorporated into nicotine must involve C-6 since nicotinic acid-G- H was incorporated with loss of the label, whereas nicotinic acid-2-3H was incorporated with retention of the label 140). A... 

The final step in the synthesis was the global deprotection. Removal of all the Z groups on the side chain amino groups of the lysine residues as well as the cleavage of the terminal benzyl ester was acconq>lished by catalytic hydrogenation over palladium in acidic solution. The use of TFA/ACOH/H2O solvent mixture gave the best results. The deprotection was con )lete in about 3h and the crude product was isolated after filtration to remove the catalyst and evaporation of the solvent. It is noteworthy that the removal of the catalyst should be done in the absence of air (oxygen) to eliminate, or minimize, contamination of the product with palladium. The pure product was isolated by prep HPLC as the acetate salt. 

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