Radioactive amino acid incorporation

Radioactive Amino Acid Incorporation. For analysis of in vivo protein synthesis in leaves of pea seedlings and of A. hybridus, a surface application of 100-150 uCi S-methionine (New England Nuclear, 1200-1400 mCi/umol) in a 100 jil solution containing 0.1% Tween-80 was performed. Uptake and incorporation of the radiolabel into protein was allowed to proceed for 4 h prior to isolation of the thylakoid membranes. 

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. 

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. 

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. 

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.

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... 

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 ... 

With the aid of the electron microscope, ribosomes were first discovered as small, discrete, RNA-rlch particles in cells that secrete large amounts of protein. However, their role in protein synthesis was not recognized until reasonably pure ribosome preparations were obtained. In vitro radiolabeling experiments with such preparations showed that radioactive amino acids first were incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains. 

EXPERIMENTAL FIGURE 20-17 The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis. The cell bodies of neurons In the sciatic nerve are located In dorsal-root ganglia. Radioactive amino acids Injected Into these ganglia In experimental animals are Incorporated Into newly synthesized proteins, which are then transported down the axon to the synapse. Animals are sacrificed at various times after Injection and the dissected sciatic nerve Is cut Into small segments for analysis with the use of gel electrophoresis. The red, blue, and purple dots represent groups of proteins that are transported down the axon at different rates, red most rapidly, purple least rapidly. 

Inactivation of certain enzyme systems involved in the oxidative metabolism of sensitive organisms by polymyxin and colistin has also been reported. This, however, might be a secondary effect . Bacitracin has been reported to interfere with cell wall synthesis. It causes Staphylococcus aureus to lyse , to form protoplasts and to accumulate cell wall precursors " . The incorporation of radioactive amino acids into cell wall mucopeptides is inhibited. Bacitracin has further been found to prevent Staphylococcus aureus from synthesizing /3-galactosidase , yet it does not interfere with the incorporation of radioactive lysine into cells . In experiments with Staphylococcus aureus, bacitracin and penicillin were shown to share a common binding site on the membrane , a result which could not be confirmed in similar experiments with B. megateriumP . Recently a direct effect of bacitracin on the cytoplasmic membrane has been demonstrated, and it was suggested that the inhibition of cell wall synthesis could be a secondary effect . ... 

This property can be exploited with a two-color pulse chase of protein turnover in living cells, using the sequential addition of FlAsH and ReAsH to label old and newly synthesized proteins. The time course of protein turnover can be determined simply by varying the time interval between removal of the first label and the addition of the second label. Unlike traditional biochemical methods of pulse chase that use incorporation of radioactive amino acids, the two-color method allows continuous imaging of single cells and can reveal additional information about subcellular localization of protein turnover. 

The negative incorporations must be interpreted with caution, since solubility aspects and transport of the radioactive precursors to the site of biosynthesis may be extremely important 45, 47, 166). The last compound cited in Table III was incorporated into galantha-mine with no randomization (presumably via isovanillin), yet isovanillin was not utilized for alkaloid formation in either the Narcissus Deanna Durbin or in Nerine bowdenii 45, 47). Phenylalanine, an established precursor of the C-6—C-1 fragment of the alkaloids when injected into the leaves, flower stems, or bulbs, is not incorporated into alkaloids when introduced via root absorption from a hydroponic solution containing the radioactive amino acid 166). 

Several biochemical studies into the effects of novobiocin on membrane synthesis have been carried out. For example, the incorporation of radioactive glucose into the mannose fractions of the cytoplasmic membrane of M. lysodeikticus has been found to be inhibited by novobiocin [31], although similar inhibitions occur in other cellular fractions. With the use of protoplasts of B. megaterium, it has been shown [67] that the incorporation of radioactive amino acids and glycerol into both membrane and cytoplasmic fractions is inhibited by similar degrees. It was concluded that, although the membrane may be malfunctional, the cytoplasmic structure of novobiocin-treated protoplasts can be similarly described. 

As previously mentioned, neither the incorporation of radioactive amino acids into embryo proteins nor the presence of amino acids in the immediate environment of the embryo necessarily indicate that free amino acids contribute significantly to the nutritional needs of the embryo. Nevertheless, it should be noted that Mintz (1964), employing autoradiography, observed in the cultured mouse embryo an increase in amino acid incorporation from fertilization to the blastocyst stage. This observation was essentially confirmed (Brinster, 1971) for the mouse when quantitative procedures for the analysis of radioactivity were used. During cleavage of the rabbit embryo in vitro (Manes and Daniel, 1969),... 

If radioactive amino acids are incorporated into mitochondrial proteins in vitro radioactivity is associated with mitoribosomes (Ashwell and Work, 1970). But this activity can be removed by puromycin treatment, suggesting that the radioactivity is the result of incomplete peptide chains on the mitoribosomes. There is no evidence that any mitochondrial ribosomal proteins are labeled in vitro although a clear-cut separation of the ribosomal proteins from animal cells has not been reported. Even with N. crassa no evidence was found that ribosomal proteins are coded for by mt DNA (Lizardi and Luck, 1972). From all these data it seems very likely that mitochondrial proteins are synthesized outside the mitochondria, under control of the nuclear genome, and are subsequently transported into the mitochondrial compartment. 

Incorporation of labeled amino acids into proteins certainly does not prove that protein synthesis takes place. In fact, denatured mitochondrial proteins bind considerable amounts of radioactively labeled amino acids and specific binding of amino acids ( transpeptidation ) to mitochondrial protein-lipid structures has been reported (Hochberg et al., 1972). Thus credit for measuring mitochondrial protein synthesis can only be given if the data include reasonable zero time controls and if the amino acid incorporation reaction is sensitive to chloramphenicol or to another suitable antibiotics known to inhibit mitochondrial protein synthesis. 

The 50 particle incorporates radioactive amino acids before these are found in complete virions, and 50 particles accumulate when RNA synthesis (and virus production) are inhibited by cordycepin. 

In an experiment designed to follow the synthesis and secretion of zymogen, a radioactive amino acid was Injected into the pancreas of a guinea pig to label proteins undergoing synthesis (pulse). Since most proteins in pancreas are synthesized as zymogens, the radioactive amino acid would be incorporated primarily in gmiogens. Three minutes after the pulse, all incorporated radioactivity was found to be present in the rough endoplasmic reticulum (ER). 

---