Synthesis reticulocytes

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

Hydroxyurea is a ribonucleotide reductase inhibitor that prevents DNA synthesis and traditionally has been used in chemotherapy regimens. Studies in the 1990s also found that hydroxyurea increases HbF levels as well as increasing the number of HbF-containing reticulocytes and intracellular HbF. Other beneficial effects of hydroxyurea include antioxidant properties, reduction of neutrophils and monocytes, increased intracellular water content leading to increased red cell deformability, decreased red cell adhesion to endothelium, and increased levels of nitric oxide, which is a regulator involved in physiologic disturbances.22... 

Waxman HS, Rabinowitz M. 1966. Control of reticulocyte polyribosome content and hemoglobin synthesis by heme. Biochim Biophys Acta 129 369-379. 

Farrell, P. J., Balkow, K., Hunt, T., Jackson, R. J., andTrachsel, H. (1977). Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis. Cell 11, 187-200. 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Obrig, T. G., Culp, W. J., McKeehan, W. L., and Hardesty, B. (1971). The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J. Biol. Chem. 246, 174-181. 

Most frequently, extracts of either prokaryotic or eukaryotic origin as such from Escherichia coli, wheat germ or rabbit reticulocytes are employed for cost reasons and availability. While those based on E. coli are unable of post-translational protein modification, eukaryotic extracts do allow synthesis of glycosylated or phosphorylated proteins to some extent when additional components, such as microsomes for glycosylation are added. Care needs to be taken with cell-free systems recombinated from the individual components when a native protein is to be produced that does not fold spontaneously... 

During p-globin synthesis in normal reticulocytes the sequoice his-atg-pro occurs at position 165-167. How many high-energy phosphate bonds are required to insert these 3 amino acids into the p-globin polypeptide during translation ... 

Accumulation of heme in reticulocytes can regulate globin synthesis by indirectly inacti-vati ng eIF-2. Which of the following steps is most directly affected by this mechanism ... 

Protein synthesis inhibition. Chromatographic fraction of the dried seed, in cell culture, was active on reticulocyte lysate of rabbits, inhibitory concentrationjf, 15.25 ng/ mL vo84. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

A well studied example for control at the level of eIF-2 is the regulation of protein biosynthesis in erythroid cells (review Chen and London, 1995). A decrease in the heme concentration in reticulocytes leads to inhibition of globin synthesis at the level... 

Similar evaluations employing an in vitro reticulocyte assay system failed to provide evidence of diminished polypeptide formation (unpub. obser.). Thus, a consistent pattern emerges in these three systems canavanine does not impede protein synthesis, including aberrant, canavanyl proteins. 

Additional information <1-7, 11, 14, 15, 17-19, 21, 30> (<7> cell-free synthesis in mRNA-dependent rabbit reticulocyte lysate system [40] <2,4,5> high activities in tissues where turnover of energy from adenine nucleotides is great, e. g. muscle [3] <1-6,11,14,15> tissue distribution [3,46] <2,5> rabbit and human carry a minimum of 2 sets of isozymes within an individual one set in muscle, erythrocytes, brain and another in liver, kidney and spleen [3]) [3, 40, 46]... 

The regulation of translation through the phosphorylation of eIF-2 is best understood as it operates in the rabbit reticulocyte. Two protein kinases specific for the a subunit of eIF-2 have been purified from reticulocytes. One of these kinases, termed the heme-regulated inhibitor repressor (HRI), serves to coordinate the rate of hemoglobin synthesis (more than 90% of the total protein synthesized in the reticulocyte is hemoglobin) with the availability of hemin (the... 

Regulation of protein synthesis in the rabbit reticulocyte. The vast majority of the protein synthesized in the rabbit reticulocyte is hemoglobin. The gross rate of protein synthesis in the reticulocyte is controlled indirectly by the concentration of heme. Heme inactivates a kinase that would otherwise inactivate the initiation complex involving eIF-2 and eIF-2B. The kinase phosphorylates the eIF-2 factor, making it impossible for the eIF-2-eIF-2B complex to exchange GDP for GTP. 

The uptake of iron from transferrin into cells has been best studied for reticulocytes, where there is heavy demand for iron for the synthesis of heme. Reticulocytes and, by analogy, other mammalian cells have receptor molecules on their plasma surface which bind transferrin. Characterization of the transferrin receptor from rabbit reticulocytes suggests that it is probably a glycoprotein of molecular weight around 200 000. The number of receptor sites in cells varies in response to the conditions. For example, treatment of K562 cells with the efficient iron chelator desferrioxamine results in an increase in the total number of receptors for transferrin.1142... 

Stavnezer, J. Huang, R.C.C. (1971). Synthesis of a mouse immunoglobulin light chain in a rabbit reticulocyte cell-free system. Nature New Biol. 230,172-176. 

Heme synthesis is controlled primarily by 8-aminolevulinate synthase (ALA synthase). There are two mechanisms of control, and each involves a process that affects the concentration of the enzyme. First, the half-life of ALA synthase, as shown by experiments in rat liver, is very short (60-70 min). Like many mitochondrial proteins, ALA synthase is encoded by nuclear genes, synthesized on cytoplasmic ribosomes, and the enzyme is translocated into the mitochondria. The second and main regulating factor is the inhibition of ALA synthase by hemin. Hemin differs from heme in that the Fe atom is in the Fe3+ oxidation state. Heme spontaneously oxidizes to hemin when there is no globin to form hemoglobin. Hemin serves a second function in the regulation of hemoglobin synthesis in reticulocytes. It controls the synthesis of globin. 

Globin is synthesized in reticulocytes (see Chap. 1, Prob. 1.1). which have no nucleus and therefore cannot utilize transcriptional and other potential modes of control. Control of globin synthesis from the pool of globin-enriched mRNA is geared to the concentration of hemin (Fe(III)-protoporphyrin]. which has the ability to inactivate a translational inhibitor of protein synthesis. The inhibitor is a protein kinase that phosphorylates and inactivates one of the initiation factors involved in initiation of translation. When the concentration of hemin is high, it binds to a regulatory subunit of the kinase and. as a result, initiation of globin synthesis can proceed. 

Transcriptional control of globin synthesis in reticulocytes is not possible because transcription does not occur in these cells. Does this mean that the overall control of globin synthesis is completely lacking an aspect of transcriptional control ... 

Incubate the reticulocytes with [3H]leucine, which will be incorporated into proteins. Prepare electron microscope autoradiographs and count silver grains per cell and the number of polysomes. The latter appear as rosettes of five ribosomes in these cells. A statistical comparison between the number of polysomes and the amount of protein synthesized during the incubation time (proportional to the number of silver grains) indicates whether there are nonactive polysomes. In fact, many of the polysomes are inactive i.e., they are switched off (see Chap. 17 for the control of protein synthesis). 

RNAP, DNA-dependent RNA polymerase RNAS, RNA synthesis ROS, reactive oxygen species rRIP, recombinant RIP RRL, rabbit reticulocyte lysate (for in vitro PSI measurement) rRNA, ribosomal RNA RT, reverse transcriptase RTK, receptor tyrosine kinase RY-R, ryanodine receptor,... 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

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