Rabbit reticulocyte protein synthesis

Kemper WM, Berry KW, Merrick WC (1976) Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. J Biol Chem 251 5551-5557 Kim SC, Sprung R, Chen Y et al (2006a) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23 607-618 Kim YS, Kang KR, Wolff EC d al (2006b) Deoxyhypusine hydroxylase is a Fe(ll)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected]. J Biol Chem 281 13217—13225... 

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-... 

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. 

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... 

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). 

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. 

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.

Some members of the human RNase A superfamily of proteins are known to have host defense activities (reviewed in ref. 9). These include, for example, two of the eosinophil cytotoxic granular proteins, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) (10). Angiogenin, a protein 65 % homologous to pancreatic RNase (11,12) that was originally isolated on the basis of its angiogenic activity (13), is a potent inhibitor of protein synthesis in the rabbit reticulocyte lysate (14) and when injected into Xenopus oocytes (15). We have therefore sought to fuse RNases to MAbs to evaluate their usefulness as immunotoxins (16,17). 

TRANSLATIONAL CONTROL Eukaryotic cells can respond to various stimuli (e.g., heat shock, viral infections, and cell cycle phase changes) by selectively altering protein synthesis. The covalent modification of several translation factors (nonribosomal proteins that assist in the translation process) has been observed to alter the overall protein synthesis rate and/or enhance the translation of specific mRNAs. For example, the phosphorylation of the protein eIF-2 affects the rate of hemoglobin synthesis in rabbit reticulocytes (immature red blood cells). 

Germination of Sphaerotheca fuliginea conidia was inhibited at rather low concentrations of the compound and when germ tubes were formed they showed spherical or oval shaped alterations [36]. Regarding the mode of action, mildiomycin interferes in protein synthesis at low concentration (0.02 mM) the compound inhibited incorporation of amino acids into polypeptides in a cell-free system of E. coli, whereas synthesis of polypeptides in mammalian cell-free systems from rabbit reticulocytes proved to be less sensitive to mildiomycin [37]. 

Not surprisingly, the toxin s liver toxicity is reflected by the ability of micromolar concentrations of cylindrospermopsin to kill in vitro liver cells such as rat hepatocytes and the human hepatoblastoma cell line HEP-G2. Cylindrospermopsin is known to potently inhibit cellular protein synthesis that can be measured in vitro using rabbit reticulocyte lysate. 

The rabbit reticulocyte, which synthesizes about 90 of its protein as hemoglobin, offers one of the best examples of translational control of protein synthesis in animal cells. In the complete absence of a nucleus (which is extruded from the cell during its maturation) it coordinates the production of the a and R chains of globin, both with each other and with the supply of heme. However, the exact mechanism of this translational control is not yet fully understood, and only a partial description of what appears to be a surprisingly complex process can be given below. 

In this section we shall present evidence that indeed eRF is a component of the system which regulates protein synthesis in rabbit reticulocyte lysate. First of all we will focus attention on the formation of the ternary complex because protein factors influencing the activity of eIF-2 in ternary complex formation have been described. Two factors, called ESP (5) and (3o-eIF-2A (6, 7) enhance the activity of eIF-2 in ternary complex formation two other factors, Co-eIF-2B (8, 9) and Co-eIF-2C (9) seem to modulate the effect of Mg on this reaction. Co-eIF-2B promotes the dissociation of the ternary complex at high Mg + concentration, >1 mM (8, 9). Co-eIF-2C, on the other hand, prevents this dissociation and appears to counteract Co-eIF-2B (8). In addition it was shown that eIF-2, phosphorylated by HRI, was irresponsive to the above described effects (5, 10). 

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

It is, of course, legitimate to argue that these mRNPs, and the free a-chain mENP of rabbit reticulocytes, represent a special class of free mRNP different from those in duck reticulocytes, but the results should at least caution against the extreme view that all mRNPs are imtranslatable in cell-free protein synthesis systems. 

The association of ( S) methionyl-tRRA with the 4O-S subunit was strongly inhibited by poly(l) poly(c) in extracts of interferon-treated cells. In extracts of control cells poly(l) poly(c) had no significant effect upon the formation of methionyl-tRHA 40-S subunit complexes. Addition of crude rabbit reticulocyte initiation factors greatly reduced the poly(l) poly(c)-induced inhibition of (55s) methionyl-tRRA 4O-S subunit complex formation. The effect was most marked when the initiation factors were added during the incubation with dsRRA, strongly suggesting that in interferon-treated cell extracts dsRHA acts at the level of a protein synthesis initiation factor which binds methionyl-tRNA to native 4O-S ribosomal subunits, a situation analogous to that of the rabbit reticulocyte lysate (20-22 and chapter 10 of this volume). 

In reticulocytes, 80% of protein synthesis is directed toward hemoglobin. Consequently, one might expect that hemoglobin messengers would be abundant and relatively easy to purify from rabbit reticulocytes. 

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