Microsomes amino acid incorporation enzymes

We have studied amino acid incorporation into mitochondrial proteins (CAP-sensitive and CH-insensitive) in vitro during late organogenesis in rats (days 11-14 of gestation). Because of microsomal contamination our data were based on the activity of a mitochondrial marker enzyme, cytochrome oxidase, (Table XIII). Protein synthesis in mitochondria... 

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). 

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. 

Intact mitochondria, like nuclei but unlike microsomes, do not seem to be affected by ribonuclease (160, 276) and may, in fact, be stimulated severalfold (276). Some workers (127,279) have claimed a partial inhibition by the enzyme, but it seems likely that this is observed only when the mitochondria have been sli tly damaged. The enzyme has been shown not to penetrate the intact structures (280) but, on the other hand, to inhibit completely the amino acid incorporation of disrupted mitochondria (280,281). 

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. 

Amino acid incorporation in isolated subcellular structures other than microsomes has been studied in much less detail than that in the latter, and with much less attention to mechanism. In no case has there been a demonstration for an absolute requirement for the pH 5 enzymes, and although in some cases some stimulation of amino acid incorporation... 

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. 

The time-dependent incorporation of amino acid into ribosomal protein is shown in Figure 2. When the crude pH 5 Supernatant fraction was used, incorporation was very rapid and essentially complete in 2 to 4 minutes. Incorporation was usually slower when the more purified enzyme fractions, transferases I and II, were used incorporation was complete after approximately 20 minutes. A similar rate of incorporation was observed when the transferase n used was isolated either from the microsomes or the post-microsomal supernatant. 

Recently, it has been reported that coumarin and some closely related derivatives administered in vivo stimulated the incorporation of labelled amino acids in the liver [223, 224] the endogenous and poly U-directed microsomal protein synthesis were also increased [225]. The relation of this increased de novo protein synthesis to the induction of drug metabolising enzymes is not clear as coumarin was found to inhibit drug metabolism [66]. However, coumarin brought about a decreased incorporation of amino acids by liver slices in vitro [226]. 

Similar in vivo labeling studies contributed other important information they demonstrated that the rate of protein synthesis was different in different tissues. Although the proteins of liver, kidney, intestinal mucosa, spleen, pancreas, bone marrow, and testicles are rapidly labeled, labeling is slow in muscle, brain, erythrocyte, and skin. Within a given tissue, some proteins are labeled faster than others. In the pancreas, the hydrolytic enzymes incorporate amino acids more rapidly than other proteins. The label appears sooner in some cell fractions than in others. After in vivo administration of antigens, 50% of the antibodies can be recovered in the microsomal fraction only a few minutes after injection. It was also shown that under conditions of net protein synthesis, labeled amino acids injected in mice are first recovered in the microsomes, then in the supernatant, and later in the zymogen granules of the pancreas. 

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. 

At least three stages are now believed to be involved in protein synthesis in animal cells. In stage (j), amino acids (AA) are activated by the formation of amino acyl adenylates in the presence of appropriate activating enzymes (E). In stage (2), the activated amino acid reacts with ribonucleic acids of relatively low molecular weight, known as soluble (or transfer) ribonucleic acids (sRNA). In stage (j), the amino acid is transferred to the RNA of the microsomes (Ms) where it is incorporated into new protein ... 

Progress has been made in demonstrating synthesis of specific proteins by cell-free systems, e.g., on the synthesis of cytochrome c by isolated rat liver mitochondria (S3, cf. 383) and on the synthesis of serum albumin by the isolated microsome fraction of rat liver 34, cf. 335,336). Campbell et al. 34) concluded that while specific serum albumin is, indeed, synthesized on the ribonucleoprotein particle fraction of the microsomes, it is not readily released in soluble form. In other words, the isolated microsomes have lost their ability to promote substrate turnover. The same is true for the hemoglobin-synthesizing RNP particles from rabbit reticulocytes 35, cf. 138). Ogata and associates 36) have essentially confirmed the results of Campbell et al. 34) on the synthesis of serum albumin by liver microsomes they have also studied the relative effect of both stimulatory and inhibitory factors on the incorporation of different amino acids into total ribonucleoprotein and into serum albumin, and showed that the requirements for the two processes were generally the same it may be noted, however, that pretreatment of the pH 5 enzymes with ribonuclease, which caused a 95% inhibition of the ineorporation into ribonucleoprotein, inhibited the corresponding incorporation into serum albumin by only 55%. 

Protein synthesis in mitochondria is dependent on the suppty of ATR either oxidative phosphorylation, or a steady supply of ATP must be provided. From a pharmacological standpoint, it is interesting that the incorporation of amino acids is affected by th3iroid hormone in vivo. The labelled amino acids are incorporated into an insoluble protein fraction present in the membrane and none of the soluble mitochondrial enzymes studied so far become labelled to any appreciable extent. The process of protein synthesis in mitochondria, as monitored by the incorporation of amino acids, displays some peculiar characteristics it is inhibited by a variety of other amino acids, possibly due to competitive effects among different amino acids for a common transport mechanism. Also peculiar is the sensitivity to chloramphenicol, and the insensitivity to cycloheximide, which is typical of bacterial systems, and not of microsomal systems. Then, there is the observation that actinomycin-D (a known inhibitor of the nuclear DNA-dependent RNA polymerase), inhibits protein synthesis in mitochondria after treatments have been applied which affect the permeability of the membrane, thus permitting penetration of the antibiotic. This last observation indicates synthesis of messenger RNA in mitochondria via a specific DNA-dependent RNA polymerase. Protdn synthesis in mitochondria is thus apparently dependent on the continuous synthesis of RNA this is possibly due to a peculiar lability of mitochondrial messenger RNA. 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

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