Amino acid subcellular incorporation

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. 

When protein synthesis is expressed in fractional terms there is no indication of the contribution that the different subcellular components make to the total liver synthesis. However, the quantities of protein in each fraction did not differ significantly at any time before or after feeding. Expressing the results in absolute terms reveals that soluble proteins in the post-mitochondrial fraction have the highest rates of synthesis and all the fractions increase with feeding (Fig. 7). It should be noted that all these experiments were carried out with labelled phenylalanine incorporation over 40-min periods and our results indicate that newly synthesised export proteins, e.g. albumin, do not appear in the fishes plasma until at least 1 h after radiolabelled amino acid injection. 

The subcellular organelle concerned with protein synthesis is the ribosome. This consists of two subunits, composed of RNA with a variety of associated proteins. The ribosome permits the binding of the anticodon region of amino acyl tRNA to the codon on mRNA, and aligns the amino acids for formation of peptide bonds. As shown in Figure 9.6, the ribosome binds to mRNA, and has two tRNA binding sites. One, the P site, contains the growing peptide chain, attached to tRNA, while the other, the A site, binds the next amino acyl tRNA to be incorporated into the peptide chain. 

Third, different subcellular structures incorporate amino acids at different rates. For most tissues, but not all, it is the microsomal fraction which has the most active amino acid incorporating siystem. The data suggest that all particulate structures are capable of protein nthesis. 

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. 

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... 

Actively growing or secreting tissues, such as microorganisms, young oocytes, differentiating embryos, root tips, liver, nerve, and exocrine cells, all have a high RNA content on the other hand, tissues which produce protein at a slower rate, such as heart, muscle, and kidney, for example, have a lower RNA content. Within a ven cell the rate of protein synthesis is greatest in those subcellular structures richest in RNA (cf. Section II) and in some broken cell preparations (87, 374) there seemed to be a direct correlation between RNA content, or the amount of RNA added, and ability to incorporate amino acids into protein. 

The reactions of phase 2 relate to the attachment of the bridge-carbohydrate residues to the polypeptide chain. There is evidence showing that this addition occurs while the polypeptide chain is still attached to, or perhaps still being synthesized on, the ribosomes.101-103 Thus, 14C-labeled 2-amino-2-deoxy-D-glucose, injected into the circulatory system of the rat, was incorporated into protein in the ribosomes of the rough endoplasmic-reticulum of the liver. Administration of puromycin caused release of the 14C-labeled glycoprotein, which could be isolated by acid-precipitation methods. Examination of the radioactivity data revealed that the subcellular structures most actively involved in glycoprotein synthesis were the ribosomes bound to the membrane, and not free polysomes. 

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