Microsomes amino acid transfer

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. 

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

Table II. Transfer of Various sRNA-Bound Amino Acids to Microsomal Proteins...

Table n) in the process of purification of the transferring factor, has suggested that this enzyme may catalyze the transfer of several or perhaps all of the sRNA-bound amino acids to microsomal proteins (10). A similar suggestion is based on the fact that the transferring activity toward several amino acids is eluted in a single peak on chromatography on DEAE-cellulose (20). 

The transfer of labeled amino acids from aminoacyl sRNA to purified rat-liver ribonucleoprotein particles has been shown to require GTP, and a soluble portion (pH 5 Supernatant) of the cell. An enzyme fraction, aminoacyl transferase (or polymerase) I, purified from the pH 5 Supernatant was found to catalyze the transfer of amino acid to protein with microsomes, but not with the more purified ribonucleoprotein particles (ribosomes). When transferase I was supplemented with glutathione and a microsomal extract, microsomal aminoacyl transferase (or polymerase) H, transferring activity was restored. Since the pH 5 Supernatant was active in catalyzing the transfer of amino acids from sRNA to ribosomal protein, it was concluded that both transferring activities were present in this crude fraction. Resolution of the two activities from the pH 5 Supernatant fraction was obtained by salt-fractionation procedures. Neither enzyme fraction was active when incubated individually or with glutathione, but together in the presence of... 

In ABL, an early step in apoB lipoprotein assembly shared by intestinal and liver cells is defective. The net result is near absence of all plasma apoB lipoproteins. ApoB synthesis from a mRNA transcript occurs, but its successful assembly into the mature lipoprotein particle does not. The inability to assemble apoB into lipoproteins was shown to be due to a defect in the mttp gene in affected individuals (Wetterau et al., 1992). Its translational product is an 894-amino acid, 97-kd, polypeptide that exists in the ER complexed with a 55-kd protein disulfide isomerase which is believed to maintain solubility, physiologic activity, and ER retention of the 97-kd peptide. The heterodimeric complex of the 97-kd and 55-kd subunits is referred to as microsomal triglyceride transfer protein (MTP) (Wetterau et al., 1992). 

From in vitro studies, possible reductants for Cr(VI) can range from small molecules and ions in the cytoplasm to complex membrane-bound enzyme systems in the endoplasmic reticulum. These reductants include certain amino acids and carboxylic acids, small peptides such as glutathione, components of electron transport systems in both the mitochondria and the microsomes, and proteins such as hemoglobin which while not normally functioning as electron transfer agents do contain redox sites. 

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 ... 

It was shown concomitantly with the discovery of sRNA by Hoagland ef al. (18S), and has since been confirmed by a munber of workers ISO, 148, 186, 188, 197, S19) that the labeled amino acid can be transferred in vibro from the sRNA to microsomes. Figure 8, taken from a paper of Hoa and et al. 186) shows the time curve of the transfer of labeled leucine from prelabeled pH 5 fraction from rat Uver to unlabeled microsome protein. All workers agree that this step requires GTP. [According to Hoagland et al. 186), in the absence of GTP there is an equally rapid microsome-dependent loss of C Meucine from RNA, but the label does not appear in... 

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). 

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. 

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