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14C-labeled amino acid

Bravo R, Cells JE. (1982) Up-dated catalogue of HeLa cell proteins percentages and characteristics of the major cell polypeptides labeled with a mixture of 16 14C-labeled amino acids. Clin Chem 28, 766-81. [Pg.153]

The ability of a tumor cell to manufacture proteins is a result of intact DNA, RNA and biochemical intracellular mechanisms. Interference with any one of these structures or processes will result in the inability of the cell to produce required proteins. Hence, quantitation of tumor cell protein synthesis over a period of time may constitute a marker allowing determination of the efficacy of a macromolecular drug conjugate. The technique is based on the fact that decreased cell viability in the presence of radiolabeled amino adds correlates to a decrease in radioactivity relative to a control cell population. For example, 3H-leucine [175, 208], a mixture of [14C]-labeled amino acids [205], and 75Se-lenomethionine [54, 209] have been used to evaluate the activity of conjugates. [Pg.90]

Ribosomes were discovered by electron micros-copists examining the structure of the endoplasmic reticulum using ultrathin sectioning techniques. Their presence in cells was established by 1956, and the name ribosome was proposed in 1957. At first it was difficult to study protein synthesis in vitro using isolated ribosomes. No net synthesis could be detected until Hoagland and associates measured the rate of incorporation of 14C-labeled amino acids into protein.26 This sensitive method permitted measurement of very small amounts of protein synthesis in cell-free preparations from rat liver and paved the way toward studies with ribosomes themselves. [Pg.1474]

In another set of experiments, Schlesinger and Anderson (44) showed that the isozymes are formed in vivo by alteration of the dimer. Using an E. coli mutant that makes an altered subunit, which will only dimerize (in vitro or in vivo) in the presence of phosphate or zinc, they found that the monomers produced by the cell growing in the absence of phosphate and zinc produced only one electrophoretic form when the monomer was converted to the dimer in vitro. However, if the medium is made 2 vaM in phosphate and 10 /iM in zinc, in the exponential phase of growth, three isozymes are formed. Additional support for the conclusion that isozymes are made by alteration of the dimer comes from the fact that independent of when 14C-labeled amino acids are added to the growing culture, label appears first in isozyme I. It thus appears that a mechanism is available in the periplasmic space for the conversion of isozyme I to the other isozymes. [Pg.386]

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. [Pg.154]

The Wolff rearrangement and the Arndt-Eistert homologation sequence are very useful in organic synthesis. One of the most popular applications involves amino acids. An interesting example has been described as a key reaction in the synthesis of a 14C-labeled amino acid used for deciphering the biosynthesis of penicillin N from glutamic acid (Scheme 3.2).9 This rearrangement proceeds without racemization and can thus be applied in peptide synthesis. [Pg.84]

The biosynthetic study of VI using 14C-labeled amino acids and growing cells of a BLM-producing strain showed that 0-alanine and L-cysteine were incorporated in situ, but VI itself was not incorporated into BLM. These experimental results together with isolation of P-SBm, of which the C-terminal is 0-alanine, suggests that VI is formed by stepwise incorporation of one mole of 0-alanine and two moles of cysteine followed by dehydrative cyclization and dehydrogenation. [Pg.89]

Unfortunately, the OPA condensation products decompose to some extent on the HPLC column (as shown by means of 14C-labelled amino acids - the positions of emerging samples shown by their fluorescence lag behind maximum radioactivity profiles), although users favouring these OPA derivatives have learned to practise strict protocols for their use, in order to get reliable results. The OPA-TV-acetyl-L-cysteine condensation product is a diastereoisomer mixture when formed with a... [Pg.58]

Methyl-, 2,4-dimethyl-, 2-ethyl-4-methyl-, 2-hydroxymethyl-4-methyl-, and 2-carbamoyl-4-methyl-quinazoline were isolated from Pseudomonas aeruginosa. (S)-Tryptophan was shown to be the precursor of these quinazo-lines by using the 14C-labeled amino acid in a new pathway of tryptophan biosynthesis73 (see Section III,A). [Pg.56]

Highly purified prostatic acid phosphatase labeled with 14C has been obtained by incubation of slices of hypertrophic human gland with labeled amino acids 53). [Pg.467]

Concentrations, turnover, and uptake of free amino acids probably have been examined more extensively than any other DOM component. For several decades it has been possible to measure turnover of dissolved free amino acids (DFAA) from the uptake of 14C- or3H-labeled amino acids (e.g., Azam and Holm-Hansen, 1973). Equally important was the development... [Pg.218]

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,... [Pg.699]

Another important technique was based on the observation that synthetic trinucleotides induced the binding to ribosomes of tRNA molecules that were "charged" with their specific amino acids 38/39 For example, the trinucleotides UpUpU and ApApA stimulated the binding to ribosomes of 14C-labeled phenylalanyl-tRNA and lysyl-tRNA, respectively. The corresponding dinucleotides had no effect, an observation that not only verified the two codons but also provided direct evidence for the triplet nature of the genetic code. Another powerful approach was the use of artificial RNA polymers, synthesized by combined chemical and enzymatic approaches.40 For example, the polynucleotide CUCUCUCUCU led to the synthesis by ribosomes of a regular alternating polypeptide of leucine and serine. [Pg.1475]

Figure 6.4 Experimental curves for equilibrium gel filtration, (a) The 1-mL tuberculin syringe was incubated in 109-pAf[14C]valine (O) or 109-/zM[14C]valine and 4-mM ATP ( ). Then 100 fjL of a solution of 26-fiM valyl-tRNA synthetase was added to the same solution. Stoichiometries of 0.8 and 1.1, respectively, were found for the binding of the amino acid. Note the return to baseline between the peak and the trough— the mark of a good equilibrium gel filtration experiment, (b) An artifact-induced double peak obtained from the binding of [y-32P]ATP and valine to the enzyme. Some of the labeled ATP hydrolyzed to [32P]orthophosphate, which traveled down the column faster than the fy-32P]ATP did. Figure 6.4 Experimental curves for equilibrium gel filtration, (a) The 1-mL tuberculin syringe was incubated in 109-pAf[14C]valine (O) or 109-/zM[14C]valine and 4-mM ATP ( ). Then 100 fjL of a solution of 26-fiM valyl-tRNA synthetase was added to the same solution. Stoichiometries of 0.8 and 1.1, respectively, were found for the binding of the amino acid. Note the return to baseline between the peak and the trough— the mark of a good equilibrium gel filtration experiment, (b) An artifact-induced double peak obtained from the binding of [y-32P]ATP and valine to the enzyme. Some of the labeled ATP hydrolyzed to [32P]orthophosphate, which traveled down the column faster than the fy-32P]ATP did.
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. [Pg.256]

Schlesinger and Anderson (44) separated the isozymes on starch-gel and DEAE-cellulose at various times after 14C-amino acids were added to a culture of E. coli synthesizing alkaline phosphatase. They found that initially most of the counts appeared in isozyme I, but later they appeared mainly in isozymes II and III. In another experiment where only enough radioactive amino acid was added to allow for 5 min of synthesis of radioactive protein, they found that the label is initially found in isozymes I and II and later in isozymes II and III. These experiments establish that the monomers of isozyme I are precursors for isozymes II and III. [Pg.386]

Nicotine.—The pyrrolidine ring of nicotine (6) derives from ornithine (1), label from, e.g., C-2 appearing equally spread over C-2 and C-5. This symmetrical incorporation of the precursor amino-acid is accounted for by the intermediacy of the symmetrical diamine putrescine (4), which is supported by other evidence too.1,2 The symmetrical incorporation of ornithine into nicotine (6) and into nornicotine (7) has been confirmed by the results4 of experiments with [2,3-13C2]ornithine [as (1)], thus reinforcing earlier 14C and 13C results (cf. Vol. 8, p. 5 Vol. 10, p. 14). Equal labelling of C-2, C-3 and of C-4, C-5 was observed. [Pg.1]


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See also in sourсe #XX -- [ Pg.84 ]




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