Radioactive Amino Acids

With the postcolumn denvatization technique, a split of the effluent before derivatization enables easy measurement of the radioactive undenvatized amino acid The precolumn derivatization technique with OPA may be equally simple, but some aspects need to be considered and examined. 

The instability and chemical conversion of some OPA derivatives imply that a denvatized compound may, in fact, result in one fluorescent and two radioactive peaks (Simson and Johnson, 1976, Fig. 1). The chemical rearrangement of the derivatives may, however, be a minor factor with respect to retention and the fluorescent and nonfluorescent derivatives may coelute. The use of more chemically stable amino acid derivatives, i.e. those formed by reaction with FMOC chloride, eliminates this problem. When the radioactivity of an amino acid is measured, it is often desirable and necessary to inject larger concentrations of amino acids than in a routine expenment. With the OPA method it is then critical to (a) make sure that OPA is present in the required molar excess (Lindroth and Mopper, 1979), (b) lower the pH of the reagent mixture to spare the top of the column, and (c) use the same or lower proportion of organic solvent in the sample as in the beginning of the gradient in order to obtain a concentration of the derivatives on the column top. 

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Metabolic labeling ofproteins with radioactive amino acids... 

Mans, R. J., and Novelh, G. D. (1960). A convenient, rapid, and sensitive method for measuring the incorporation of radioactive amino acids into protein. Biochem. Biophys. Res. Commun. 3, 540-543. 

Three major advances set the stage for our present knowledge of protein biosynthesis. First, in the early 1950s, Paul Zamecnik and his colleagues designed a set of experiments to investigate where in the cell proteins are synthesized. They injected radioactive amino acids into rats and, at different time intervals after the injec-... 

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. 

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,... 

Radioactive amino acids are commonly used to follow protein synthesis. The procedure is in general similar to that used to follow synthesis of DNA, but in this case the cell s growth medium already contains high concentrations of amino acids and so the added radioactive amino acid acts as a tracer without causing imbalance. However, when it is desired to increase the extent of radioactive incorporation the presence of amino acids in the growth medium may be a disadvantage and their concentration may be reduced. Drastic reduction is obviously deleterious to cell growth ( 11.7] and preliminary experiments must be performed to ascertain what reduction can. be tolerated under the experimental conditions. 

The experiments discussed were performed with Lactobacillus arabinosus using glutamic acid, alanine, and proline as the test amino acids. The detailed experimental procedures have been described (21, 23, 27). Washed resting cells are incubated in a phosphate buffer with the radioactive amino acid, centrifuged, and extracted to release the amino acid, which is then measured enzymically or by isotopic methods. 

Protoplasts of C. purpurea have been prepared which are able to synthesize the peptidic ergot alkaloids ergotamine (69) and ergocryptine de novo.63 Various radioactive amino-acids were incorporated into the alkaloids, and D-lysergic acid (70) stimulated their utilization. It was the only precursor to stimulate the synthesis of alkaloids, and the proposal was made that the concentration of (70) in the cells is a rate-limiting factor in alkaloid synthesis. 

The activity is usually followed by measuring the amount of RNA- AA, the product of reaction (2) formed during the incubation. Since the radiochemical detector cannot differentiate the free radioactive amino acid... 

This separation step requires first the addition to the sample of an acid such as trichloroacetic add (TCA), which also serves to terminate the enzymatic reaction. However, since TCA also precipitates the RNA and any radioactive amino acid covalently linked to it, the reaction product RNA- AA will be precipitated as well. And since the precipitate can be separated from the soluble components by a sample filtration step, the separation of the bound from the free amino acid can be accomplished. As illustrated in Figure 1.3, the reaction product, which is trapped on the filter as a precipitate, can be detected by transferring the filter to a scintillation counter for quantitation and, of course, measuring the amount of product formed. Since assays of this design usually focus on one component at a time, no information is obtained about the amount of ATP, AMP, PPj, or free amino acid during the course of the reaction. 

Figure 1. The rate of protein synthesis in soleus muscles from rats fed different levels of dietary protein. Synthesis was measured as the incorporation of a radioactive amino acid (tyrosine) into muscle proteins. Data from Ref. 16.

One of the earliest applications of immunochemical techniques in the field of molecular biology was the immunoprecipitation of an enzyme as a means of demonstrating its de novo synthesis. Such a demonstration in bacteria might involve growing the cells under two sets of physiological conditions, one in which the enzyme is expected to be present and a second in which it is not. A radioactive amino acid is added to each culture as a means of labeling newly synthesized proteins. The cells are... 

Figure 13. A composite of three drawings Model of tonofilament assembly (13A) based on the ribosome model (13B) (Ref. 79), tne distribution of radioactive amino acid uptake shown by silver grains (13C) (Ref. 74), and the cross section model (13D) Ref. (70). Second and fifth layers (dotted in 13D) are omitted from 13A. The triangles in 13D locate three possible layers of tripeptide chains.

Some phases of the synthesis of hemoglobin have been studied in vitro and in vivo in both animal and human subjects by various investigators [(G9, Hll, M4, N13) and others]. Labeled amino acids, i.e., leucine- C, phenylalanine- C, and leucine- H, are used for the study of amino acid incorporation. The procedure consists of obtaining either human or animal mRNA, reticulocyte ribosomes from the study case, and radioactive amino acids. The three are combined and incubated for... 

Liver Slices - Liver slices prepared from rats bearing an inflammatory lesion caused by subcutaneous injection of turpentine show an increased capacity for the incorporajjgn of radioactive amino acids and glucosamine into acute phase proteins. 

Experiments show that it is on the ribosomes that protein synthesis actually occurs. If cells synthesizing protein from radioactive amino acids are studied, the radioactivity is first found bound to the ribosomes, and is only later released from them as soluble protein. A cell suspension from which ribosomes have been removed can never be made to synthesize protein, whilst if they are subsequently replaced synthesis can proceed rapidly. But the ribosomes alone are inadequate. In order to incorporate radioactive amino acids into new protein there needs to be added to the ribosomes a preparation of soluble cell material which contains certain enzymes, the soluble low-molecular-weight messenger RNA (m-RNA), transfer RNA (t-RNA), ATP, GTP, and ions like magnesium and potassium. So what role do these various substances perform in protein synthesis ... 

The amount of proteins present in a large number band separated on 2D gel can be measured, and their relative abundance can be established by a variety of methods. First, the different samples of proteins are separated on 2D gel, and then the intensity of protein bands is measured by the intensity of dyes used to visualize the bands. The intensity of protein bands can be measured by a densitometric scan. Staining with silver stain is sensitive. Staining with certain fluorescent dye is equally useful. Alternatively, proteins in two cell types are labeled by growing cells in the presence of radioactive amino acids, such as methionine containing S35 sulfur. The protein samples obtained from the two cell types are then separated by 2D gel. The protein bands are visualized as spots on an X-ray film placed on the gel. The intensity of each spot on the film is determined by densitometry. 

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