Amino acid incorporated into proteins with

Anabolic activities of testosterone, such as increases in amino acid incorporation into protein and in RNA polymerase activity, have been demonstrated in skeletal muscle. Apart from the direct anabolic effects in specific tissue, androgens antagonize the protein catabolic action of glucocorticoids. The androgen compounds with the greatest ratio of protein anabolic effects to virilizing effects are the 19-nortestosterone derivatives. Compounds that are used clinically (Table 63.3) include nandrolone phenpropionate (Durabolin), nandrolone decanoate... 

All the amino acids incorporated into proteins in the body are stable under physiological conditions. However, some are easily modified by chemical or enzymatic reactions while others are difficult to change. The more reactive amino acids are those with acidic or basic groups on the side-chain (aspartate, glutamate, arginine, histidine, lysine) or sulfhydryl (Fig. 3.2) or hydroxyl groups (cysteine, serine, threonine). The more reactive amino acids are often part of enzyme active sites (Chapter 7) where they participate in the reaction catalyzed by the enzyme. 

General description. Within the first hour after infection of L-cells with mengovirus, there is an inhibition in the rate of amino acid incorporation into protein. This inhibition reaches a low point at around three hours after infection, increases slightly until about five hours after infection, and then falls (Figure I). The rate of viral protein synthesis never reaches that of the uninfected, control culture in this system. 

Studies on cell-free systems of liver are still in a preliminary stage and have yielded results difficult to reconcile with the notion that cortisone stimulates protein synthesis. These studies suggest that adrenalectomy increases and large doses of cortisone decrease amino acid incorporation into proteins. Studies in which the Zamecnik-Keller systems were used suggest that the hormonal imbalance affects the microsomes rather than the supernatant fluid of the liver. 

Adding insulin to media in which diaphragms are incubated with labeled amino acid stimulates the incorporation of the amino acid into protein. The injection of insulin to normal rats increases the incorporation of labeled amino acids into liver microsomes, and insulin stimulates the incorporation of [ " Cjglycine and [ " Cjphenylalanine into the protein of liver slices of alloxan-diabetic rats. The stimulation of amino acid incorporation into protein under the effect of insulin is independent of the effect of the hormone on glucose penetration in the cells, since it occurs when glucose is absent from the medium. The effect of insulin on protein synthesis raises the question of insulin s site of action in the sequence of steps that lead to the... 

The question raised by Schoenheimer and co-workers may be stated as two extreme alternatives (1) amino acid incorporation into proteins occurs by synthesis of protein de novo from amino acids or (2) it occurs by an enzyme-mediated exchange of amino acids in the protein with the corresponding free amino acids in the medium, without hydrolysis of the moieties on either side of the two peptide bonds split in the exchange. Some alternatives between these two extremes are that the amino acid is first incorporated into a peptide by de novo synthesis or by replacement, and the peptide is then built into a protein either by de novo synthesis of the protein or by peptide replacement. 

A major assumption underlying nearly all the work on amino acid incorporation into proteins is that radioactiAdty in the protein after incubating a tissue with a radioactive amino acid, or finding in an amino acid after an experiment with an N Mabeled amino acid, means that the labeled amino acid had been incorporated into it by peptide bonds. The whole structure of interpretation rests on this assumption. It has been rigorously proved in only two cases and the proof is almost (iomplete in only one other. 

The incorporation of labeled amino acids by isolated nuclei from calf th3onus has been studied in detail by Allfrey and co-workers 1 4), and also by Breitman and Webster 66), and by Hopkins 1 4)- Nuclei from the thymus of puppies 67), from rabbit appendix 68), from chicken kidneys l 4)t from rat liver 69), from a lymphoma 64), and from wheat germ 70) have also been studied in this respect. The incorporation of labeled amino acids into the nuclear proteins is dependent on the presence of Na+ (the microsomal system requires K+) and upon oxidative phosphorylation. No effect of added amino acids or ATP have been demonstrated, but when the nuclei lose the ability to synthesize ATP from AMP, they also lose the ability to incorporate amino acids 71)-, however, they cannot utilize AMP supplied by the incubation medium, which means that they have to be prepared so as to retain their internal nucleotide pool. Amino acid incorporation also seems to be dependent upon a preliminary synthesis of RNA, since it is completely abolished by benzimidazole derivatives which inhibit the s3mthesis of the latter. However, intact RNA does not seem to be needed, and treatment with ribonuclease actually enhances amino acid incorporation into protein 7 ). 

Figure 15 shows a summary of the reactions of the adenylate pathway, as we now understand them. The reliability of this information has been discussed in the previous section, and we had concluded that these reactions represent at least one of the pathways of amino acid incorporation into protein. Let us then examine this scheme with the idea of pinpointing any concrete examples of the types of relationships between RNA and protein synthesis that are most frequently observed in vim i.e., the obligatory presence of preformed RNA, the dependence of RNA synthe or turnover on the presence of amino acids, and the dependence of amino acid incorporation on some sort of metabolic activity of RNA. 

Headier and Greenberg (68) tested the two baae mechanisms put forward by Hanes et al., i.e., y-glutamyl activation is an important first step in peptide synthesis and the stabilization of 7-glutamyl peptide bonds by free amino acids thus facilitates amino acid incorporation into proteins. They compared the incorporation of labeled glycine in y-glutamylglycine with that of free-labeled glycine in the presence of... 

There are also changes in the rates of metabolism as red blood cells appear and aerobic processes intensify (Lasker and Theilacker, 1962 Laurence, 1975 Timeyko and Novikov, 1991) during the early phases of ontogenesis. Oxygen consumption increases, as do the number of mitochondria and their protein contents (Abramova and Vasilyeva, 1973 Ozemyuk, 1993). The adenyl nucleotide pool (ATP and ADP) decreases (Milman and Yurovitsky, 1973 Boulekbache, 1981), while the activity of cytochrome oxidase increases (Ozemyuk, 1993). The increased energy metabolism corresponds to a considerable extent with motor activity (Reznichenko, 1980). In the yolk sac, the activity of proteinase, which supplies nitrogenous materials to the embryo, increases, as does the rate of amino acid incorporation into the body proteins. 

Most of the experiments on incorporating amino acid esters into proteins during the plastein reaction have been carried out with papain, indicating that it is one of the best enzymes for this purpose. Other enzymes such as chymotrypsin (40) or carboxypeptidase Y from Sac-charomyces cerevisiae (41) are potent catalysts for peptide synthesis in homogeneous systems using N-acylamino acid esters of peptides as substrates and amino acid derivatives or peptides as nucleophile components. Adding organic co-solvents favored peptide bond synthesis (42,43). 

Fig. 5.1-15 Nonnatural amino acids carrying fluorescent groups, that have been incorporated into proteins with high efficiency.

A proteinase-catalyzed reaction including splitting and synthesis of peptide bonds is a process also suitable for covalent amino acid incorporation into peptide chains. This type of enzymatic modification reaction of food proteins is useful for different purposes alteration of sensory properties, solubility, nutritional quality, functional properties, antifreeze character, and different biological activities. Recently, special proteinase-catalyzed reactions have been elaborated by which proteins can be modified with particular respect to their primary structure and conformation. 

In the protein elongation process the EF-Tu and EF-G cycles themseves interact with the mRNA-pro-gramed ribosome cyclically and since (i) EF-TU has to be released from the ribosome before EF-G can bind to it and vice versa and (ii) GTP hydrolysis is required for the release of both factors, there is a stoichiometric relationship between the number of GTPs hydrolysed and the number of amino acids incorporated into the protein synthesized. This contrasts with the other GTPases, e.g. heterotrimeric G-proteins, Ras proteins, which continue to transmit a signal as long as they remain in the E - GTP form, a key feature of the signal amplification process. 

Fig. 6. Effect of triiodothyronine on amino acid incorporation and cytochrome content of mitochondrial fraction. Circles, amino acid incorporation into mitochondrial protein triangles, cytochrome as squares, cytochromes c plus Ci. Thyroidectomized animals were treated with triiodothyronine, and the results are given as percentages of the value with untreated animals. From Tata [152] Roodyn ei al. [153].

The presence of ribonuclear protein particles active in amino acid uptake has been demonstrated in nuclear preparations. The so-called nuclear ribosomes require ATP and GTP for amino acid uptake, exactly as cytoplasmic ribosomes require the same triphosphonucleo-tides for activity. There seems to be one major difference between nuclear and cytoplasmic ribosomes. RNase, which effectively blocks protein synthesis in the cytoplasm, is without effect on protein synthesis in the nucleus. The fact that DNA is required for amino acid incorporation into nuclear protein cannot be invoked as an argument in favor of the existence of such a metabolic pathway in the nucleus, because the effect of DNA is indirect and DNase interferes with the generation of ATP in the nucleus and therefore, abolishes the only source of energy in the system. 

The panel of fluorinated amino acids incorporated into peptides and proteins has seen tremendous growth in the past decade. Analogs of valine, leucine, isoleucine, methionine, alanine, and phenylalanine have been synthesized with varying numbers of fluorine atoms and incorporated into naturally occurring and de novo designed proteins and peptides. As leucine is the most widely found amino acid in proteins, trifluoroleucine and hexafluoroleucine deserve special mention as widely studied residues in peptide and protein design. 

A summary of the major findings on the incorporation of labeled amino acids into proteins is (1) every tissue that has been tested has been found to incorporate into its proteins, in vitro as well as in vivo, every common labeled amino acid (n-form) presented to it (2) so far one amino acid which is not a normal protein constituent—ethionine—has been found to be incorporated, two others—a-aminoadipic acid and a-aminobutyric acid —were not incorporated (3) the rate of incorporation varies with the amino acid and the tissue from approximately 0.1 to 10 micromoles per gram of protein per hour (4) the incorporation of a single amino acid appears to be largely independent of the presence of others except for specific accelerating effects of a few amino acids (5) labeled amino acids are incorporated by peptide linkage (6) in most but not all cases inhibitors of respiration and phosphorylation inhibit amino acid incorporation and protein synthesis (7) there is evidence that in some cases cofactors (unidentified) are involved. 

The reaction rate is affected by, among other things, the nature of the amino acid residues. Hydrophobic amino acid residues are preferably linked together (Fig. 1.51). Incorporation of amino acid esters into protein is affected by the alkyl chain length of the ester. Short-chain alkyl esters have a low rate of incorporation, while the long-chain alkyl esters have a higher rate of incorporation. This is especially important for the incorporation of amino acids with a short side chain, such as alanine (cf. Table 1.40). 

However, no further evidence for such amino acid phosphates was found, and there was no progress at all in the identification of activated precursors of protein until a fewyears ago when Hoagland et al. 94,97) found amino acid-dependent exchanges of radioactive pyrophosphate with the two terminal phosphates of ATP in the soluble liver fraction required for amino acid incorporation into microsomes. At the same time the existence of such a system in bacterial extracts was demonstrated by DeMoss and Novell 98, 99), and by Berg 100). The pyrophosphate exchange as well... 

Secondly, one wants a structure which is a good acceptor for an acyl group but a poor one for a phosphoryl group. This could be an SH, a reactive OH, or the NH of an imidazole or amide group. Among the known vitamins and cofactors there are, of course, several with the necessary structural requirements outlined above. The first to come to mind is CoA, but so far it has not been possible to demonstrate a CoA requirement for amino acid incorporation into mammalian or plant microsomes. Nevertheless this vitamin seems to be essential for the incorporation of amino acids into the proteins of hen oviducts (56) and it does, of course, the job of displacing activated fatty acids from their activating enzymes (170). Vitamin Bi2 also fits the structural requirements and it has, indeed, been claimed to be essential for amino acid activation and subsequent incorporation into rat liver microsomes (i07, 178, 179), but this requirement has not yet been confirmed by other authors (180,181). 

When unwashed particles are used, a net increase in protein is obtained by incubating them in a phosphate buffer with nothing more than Mg" and ATP (256). This increase is accompanied by the incorporation of a radioactive amino acid (phenylalanine) into protein. The detailed kinetics of both the net synthesis and the incorporation reaction vary somewhat with the state of the particles. When freshly prepared particles are used, the kinetics are deceptively simple both the amount of new protein. synthesized and the amount of phenylalanine incorporated increase fairly linearly for about 2 hr and then level off. The maximiun net increase in protein corresponds to an average of 150 ixg per milligram protein originally present, and the incorporation of C -phenylalanine to about 5 m/imoles per milligram of total protein (i.e., particulate plus newly-synthesized protein). If this is recalculated in terms of newly-synthesized protein only (since the protein originally present would presumably not incorporate amino acids), the amount incorporated is 38 m/ moles per... 

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