Lysine, radioactive

Again, Eu-labeled secondary antibodies are used, for example in an in vitro assay for the PKMT G9a [58] or for cellular hypomethylation of the target of the lysine methyl-transferase S ET7/9 [59]. An alternative is a radioactive assay, in which the biotinylated synthetic peptide substrate is bound to the cavities of avidin-coated microplates. 

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,... 

Since the glucose-lysine reaction mixture used in this study consisted of a number of different substances, it was of interest to study whether the observed inhibitory effect in vitro could be attributed to some specific compound(s). In order to obtain a separation, an aliquot of the LMW fraction, radiolabel led by [U- glucose added to the reactants, was applied on a Sephadex G-50 column and eluted with water. The UV absorbance was recorded and the eluate was collected in fractions. The degree of inhibition effected by small samples of equal volume from each fraction and exerted on carboxypeptidase A and purified aminopeptidase N was determined as well as the radioactivity... 

Thermal polylysine also catalyzes the formation of a-ketoglutaric acid from glutamic acid with CuCl25). A reaction mixture of lysine-rich proteinoid (20 mg), 14C(U)-l-glutamate (0.1 mM), and CuCl (0.1 mM) in 6 ml of Britton-Robinson buffer (pH 7.0) is incubated at 37.5 °C for 2 hours. More than 40 % of the radioactivity used is recorded in a-ketoglutaric add by paperchromatography of the reaction mixture25 . Free lysine and Leuehs poly-L-lysine have no activity 2S). The reaction obeys Michaelis-Menten kinetics at optimum pH 7.0 25). 

Selective formation of microparticles from polynucleotides and lysine-rich proteinoids rich in individual radioactive amino acid has been studied and the focus of attention is on those homoanticodonic amino acids having one homogeneous codon (glycine, CCC lysine, UUU proline, GGG and phenylalanine, AAA)58). Precipitation of individual amino acid rich proteinoids with each of homopolyribonucleotides, with and without Mg2+, was tested58>. The results show that three (Lys-rich, Gly-rich,... 

Most of the work on the biosynthesis of Lythraceae alkaloids has been done by Spenser et al. (10, 84-87). First, the validity of the pelletierine hypothesis (c) of Ferris et al. (62) has been tested. The pelletierine (126) nucleus is generated from L-lysine (181) via cadaverine (182), and presumably A -piperideine (132) and its side chain originate from the acetate. Incorporation of radioactivity from 14C-labeled samples of these precursors to decodine (6) and decinine (2) in Decodon verticilatus has been investigated (85, 87). 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

Metabolic Transit. Free Amadori Compounds. It is well known that the synthetic Amadori compounds of the free amino acids are absorbed by the intestine and excreted unchanged in the urine (9,28,30). The transport is not active as observed with deoxyfructosyltryptophan (30) and c-deoxyfructosyllysine (40), and the level of absorption depends on the nature of the amino acid and on the conditions of ingestion. Nutritional assays and metabolic transit studies performed with radioactive Amadori compounds of tryptophan (12,30), leucine (12), and lysine (9,28,41) given orally or intravenously on normal or anti-biotics-treated animals have shown that the intestinal microflora can regenerate part of the amino acid. This can be absorbed subsequently at a very low level by the caecum or the large intestine and incorporated into the tissue proteins or utilized by the intestinal microflora. Barbiroli (13) showed also that some intestinal enzymes were able to liberate some amino acids from their Amadori compounds but to a very small... 

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 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... 

Whole-Body Autoradiography. Whole-body autoradiography performed 8 h after intravenous injection of e-deoxyfructosyl-U-14C-lysine shows that the radioactivity is localized in the bladder and in the kidneys since the excretion is not complete, and a little in the pancreas (41). Twenty-four h after oral ingestion, the radioactivity is localized mainly in the large intestine and a little in the cortex of the kidneys, giving the same pattern as 14C-lysinoalanine (see Figure 2). 

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. 

We have incubated these two isopeptides with homogenates of intestinal mucosa, liver, and kidneys and showed tht c-(/ -aspartyl)lysine was not at all hydrolyzed while c-(y-glutamyl) lysine was hydrolyzed only by the kidneys homogenate. On perfused rat liver, the radioactive free e-(y-glutamyl)-14C-lysine was not transformed biochemically at all. 

Metabolic Transit of 14C-Lysinoalanine in Rats. The radioactive 14C-lysinoalanine was synthesized from uniformly labelled 14C-lysine. The compound a -N-formyl-14C-lysine, prepared according to... 

Other derivatives of lysine, c-(y-glytamyl) lysine and c-formyllysine, are hydrolyzed in the kidneys, and the lysine moiety is regenerated and incorporated into the kidney proteins in the cells where hydrolysis occurs. No retention of radioactivity occurs with derivatives such as a-formyl-14C-lysine which is not hydrolyzed in the kidneys (83) (see Figure 2). At present, we do not know yet whether these derivatives of lysine induce nephrocytomegaly. 

In the case of casein treated at pH 10 with caffeic acid, the level of the radioactivity incorporated in the liver, kidney, and muscle proteins were lower than that measured for untreated casein and casein treated at pH 7, indicating a higher loss in available lysine as expected from the high fecal excretion measured. 

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.

The quinolizidine alkaloid matrine (32), like lupanine (27), is biosynthesized from three molecules of lysine by way of cadaverine.1,2 AM6-14C]Piperideine [as (31)] was incorporated into matrine (32) 10% of the radioactivity was found to be... 

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