Microsomal system, amino acid incorporation

The aminoacyl transfer reaction, one of the latter stages in protein synthesis, involves incorporation of amino acids from soluble ribonucleic acid-amino acid into ribosomal protein. This reaction requires guanosine triphosphate and a soluble portion of the cell. Evidence has been obtained with rat liver preparations that aminoacyl transfer is catalyzed by two protein factors, aminoacyl transferases (or polymerases) I and n, which have been resolved and partially purified from the soluble fraction. Transferase n activity has also been obtained from deoxycholate-soluble extracts of microsomes. With purified transferases I and n, incorporation is observed with relatively low levels of GTP its sulfhy-dryl requirement is met by a variety of compounds. The characteristics of this purified amino acid incorporating system, in terms of dependency on the concentration of its components, are described. 

The question remains whether CAP under certain circumstances blocks protein synthesis at the 80 S ribosome. In a cell-free system of reticulocyte microsomes (Weisberger et al., 1964 Beard et oL, 1969) an inhibition of the association of mRNA and the 40 S ribosomal subunit was reported. In the reticulocyte system used 70-100% inhibition of [ Cjleucine incorporation was reported at 0.1 pmole/ml of CAP with either endogenous template RNA or small amounts of poly(U). These results could not be confirmed by Zelkowitz et al. (1968). We also found no striking inhibition of poly (U)-directed amino acid incorporation by CAP in messenger-depleted cell-free systems from rat liver or rat embryos (Uehlin et al, 1974). From all this it seems clear that generally CAP has no striking effect on protein synthesis at the 80 S ribosome level. But inhibition of mammalian microsomal protein synthesis under certain conditions cannot be ruled out. 

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. 

Although it is in the nature of kinetic experiments to be capable of disproving certain mechanisms but never to furnish positive proof, the results described in the previous section provide a rational basis for the investigation of amino acid incorporation in subcellular systems. Such investigations in the case of microsomes preceded the in vivo experiments, but in the case of nuclei, mitochondria, and bacterial membrane preparations they were a more or less direct outgrowth of the latter. In any case, as we shall see, the in vivo experiments provide a valuable point of reference for in vitro studies. 

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... 

So much work has been done, and so much emphasis has in recent years been placed on the components of the soluble cytoplasmic fractions that one might, at times, get the impression that these are studies in protein synthesis proper. In actual fact, however, the activation of amino acids and their incorporation into RNA are only preliminary steps, and the pertinence of these reactipns to protein synthesis might still be argued. No incorporation of amino acids into protein by the soluble fraction alone has ever been demonstrated, and the sine qua non of protein synthesis in the microsomal system, i.e., amino acid incorporation into protein, are the ribonucleoprotein particles. 

The system from pea seedlings, studied by Webster (260, 261) and by Raacke (256, 257, 262) differs from other microsomal systems in many important respects. Not only, as we have mentioned already, is the level of amino acid incorporation one to two orders of nu itude higher than that of other systems (see also Table X), but, more important, the label does not remain particle-bound and appears in soluble protein and, furthermore, there is a large net increase in protein, as measured by the method of Lowry at al. (263), as well as by biuret and micro-Kjeldahl. 

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 ). 

The soluble fraction of the cell, required for amino acid incorporation by microsomal systems of animal origin, enhances the activity of mitochondria, but is not absolutely essential (S65, 276, 277). There is also some disagreement among different workers as to which component of the soluble fraction is responsible for this enhancement in activity. According to some (276) the pH 5 enzymes are as effective as whole cell sap, while others (ISO, 277) find that the latter is more effective, but that the pH 5 enzymes can produce maximum enhancement if GTP is also added. Still others (150, 278) claim that boiled cell sap is as effective as fresh, and that the activity of the latter can be simulated by a mixture of amino acids and nucleotides. 

The membrane systems from the other organisms are prepared from protoplasts, and hence do not contain cell wall components. Nevertheless, they are very complex, as is perhaps illustrated best by the great differences in the level of incorporation reported for different systems—and for the same system, under different conditions. The data available from the literature are reproduced in Table XII. Despite the great variations in the reported data, it is seen that this system has a vastly greater capacity for amino acid incorporation than isolated microsomes or ribonucleoprotein particles. The only system of the latter kind which approaches the membranes in activity, is the one from pea seedlings (see Table X, Section III, B, 4, d). These two systems have other points of similarity both remain active for 2-3 hr of incubation, and the kinetics are often similarly complex. 

Let us consider the first question. Since it has been shown that amino acid-RNA compounds can be formed by highly purified amino acid activating enzymes, and the properties of this reaction and of the reaction product correspond closely to those observed in crude systems or in vivo, it seems to be established beyond reasonable doubt that the mechanism of this reaction is as outlined in Section III, B, 3, d. Furthermore, the demonstration that the amino acid bound to transfer RNA can be transferred to the microsomes and be bound there in the interior of a peptide chain, seems to show that the RNA-amino acid compound can serve as a donor of amino acid for the incorporation reaction however, since not only GTP, but also ATP and soluble fraction (which mi t contribute a large number of other factors besides the transferring enzyme) are required for the transfer, the role of sRNA-amino acid is less clear-cut than it might be. A reversal of the reaction back to the adenylates, however, and incorporation by some other route seems to be excluded by the fact that even a hundredfold excess of nonisotopic amino acid does not interfere with the efficiency of the transfer, which under the right conditions, approaches 100% (14S). The evidence to date, then, indicates that the adenylate-sRNA pathway is a pathway of amino acid incorporation in the microsomal system of mammalian origin. 

The fourth question takes us close to the core of the problem of the mechanism of protein s3uithesis, and if we knew the answer unequivocally, we could really say we knew something about the way proteins are made in vivo. Unfortunately, we do not have this information, and hence we do not know how far our knowledge of the isolated system applies. All we can say at the present time is that in mammalian systems the sRNA seems to be labeled in a manner sufficiently rapid to be consistent with a precursor role 186). The time scale employed may, however, be much too long to warrant any definitive deductions, and, furthermore, it has not been shown in vivo that the RNA is unlabeled as rapidly as it is labeled, which must be the case if it were an obligatory intermediate. Since the adenylate pathway has been studied so far only in relation to amino acid incorporation in the microsomal system in vitro, and not in relation to net protein synthesis, the ultimate answer to this question is, moreover, intimately tied to solvii the problem of the exact relationship between amino acid incorporation and protein synthesis. This will be discussed in the next section. 

Specifically, we may ask the following questions First, what constitutes amino acid incorporation into protein or protein-like material Second, is all incorporation of amino acids into protein-like material due to a de novo formation of peptide bonds Third, what are the factors which influence the apparent rate of incorporation, both in vivo and in vitro systems Fourth, what is the relationship between incorporation of amino acids into total protein-like material and the synthesis of a specific and independent protein molecule and, finally,- what is the nature of the amino acid incorporation in vitro, particularly in the microsomal system, and its relationship to amino acid incorporation in the intact cell ... 

The influence on protein synthesis of general metabolic conditions, such as malignancy, regeneration, and hormonal imbalance, has also been studied quite extensively. Each of these aspects, however, is a field in itself and cannot be reviewed here. Furthermore, most of these studies yield little information aibout the mechanism of protein synthesis. Recently, however, a few workers have started to investigate the influence of these conditions on the competence of the various components of the microsomal system to function in amino acid incorporation in vitro, an approach which promises to yield some interesting insights. Campbell and Greengard... 

The search for better cell-free preparations for studying amino acid incorporation and protein synthesis in vitro has continued, and a number of novel systems have been developed. Satake and co-workers 10) studied the incorporation of leucine into microsomal protein by a purified system from guinea pig brain, and found it to be in all respects similar to the classic microsome-pH 5 enzyme systems of rat liver and ascites tumor cells (cf. 9S, 53) (see also Table X). Other systems of this type have been obtained from the developing endosperm of maize kernels and from 2-day old maize seedlings by Mans and Rabson 11). No net synthesis of protein could be observed in either of these systems. 

It was established that the incorporation of labeled leucine reflected true synthesis and did not result from absorption or exchange of amino acids within the protein molecule. Two observations support actual protein synthesis. One is that the incorporation is energy dependent, and the other that the labeled proteins do not lose their label in the presence of large amounts of unlabeled amino acids. The cell-free system required five components microsomes, a pH 5 enzyme precipitated from the supernatant, ATP, GTP, GDP, and the labeled amino acids. 

Many investigators have studied the incorporation of labeled amino acids into proteins. The system involved in mammalian cells seems to be associated with microsomes, but requires factors from the soluble portion of the cell. Both ATP and GTP are required. The function of the ATP may be to form activated amino acids. Several systems have been found with relative specificity for various amino acids that catalyze the exchange of pyrophosphate with ATP in the presence of the amino acid, and presumably form adenyl-amino acid compounds. The activated amino acids react with hydroxylamine to form hydroxamic acids. Several reactions have been considered as models for peptide bond formation. These include the formation of hippuric acid, in which benzoyl CoA condenses with glycine in a reaction similar to the acetylation of... 

When sRNA labeled in vivo with P H21, 222) or with C -orotic acid 197) are used in the transfer system, a small amount of label is transferred to the microsomes under conditions similar to those required for the transfer of amino acid. If sRNA is terminally labeled in vitro with C -ATP or C -CTP, the label is also transferred 148)-All the experiments concerning the transfer of amino acid to microsomes are, as yet, of a rather preliminary nature, and are difficult to assess. Certainly, they permit no conclusions as to the mechanism of this reaction (or reactions). To add to the obscurity of this step, it should be pointed out that the nature of the binding of the transferred amino acid in the microsomes is not known with certainty. Webster 120) found that in three systems, pig liver, yeast, and pea seedlings, all of the transferred amino acid (which however was less than 20% of that bound to RNA) was capable of reacting with fluorodinitrobenzene, and hence was AT-terminal. Hoag-land s group 148) has shown that at least a portion of the amino acid is bound to microsomal RNA but, under conditions where the transfer is nearly 100% complete, most of the amino acid seems to be incorporated into protein 148). 

Incorporation op Labeled Amino Acids by Different Microsomal Systems... 

Although we do not know much about the mechanism of the microsomal incorporation reaction, we know quite a bit about its (empirically established) characteristics. First, from a quantitative point of view, we may ask how much amino acid is incorporated. Table X ves the maximum level of incorporation for a number of systems. Because, as we shall see, most systems continue to incorporate for very short periods of time only, the final level of incorporation is more useful in evaluating the competence of a system than the initial rate of the reaction. 

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