Reticulocyte-binding proteins

Myosin light chain 1, slow-twitch muscle b/ventricular isoform (22025.01 Da) Reticulocyte-binding protein 1, PvRBP-1 = transmembrane-anchored (325851.41 Da)... 

Reticulocyte binding protein 1 precursor-Plasmodium vivax (330217.5 Da) Titin-rabbit (fragment) (751113.32 Da)... 

Rayner, J. C., Vargas-Serrato, E., Huber, C. S., Galinski, M. R., and Barnwell, J. W. (2001). A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBPl) defines a trypsin-resistant erythrocyte invasion pathway. ]. Exp. Med. 194, 1571-1581. 

An in vitro reticulocyte translation assay (Boehringer Mannheim) was modified to determine an inhibition of translation by alkaloids. An assay (total volume 25 jl) contained 2 jl 12.5 x translation mix (Boehringer), 10 pi reticulocyte lysate, 200 mM K-acetate, 1.5 mM Mg-acetate, 0.25 pCi L-[4,5-3H(N)]-leucine, 0.5 pg TMV-RNA (Boehringer) and up to 5 mM alkaloids (buffered to pH 7). The mixture was incubated at 30 °C reactions were terminated after 0, 10, 20, 30 and 40 min. The radiolabeled protein was precipitated by adding 200 pi ice-cold trichloroacetic acid (TCA) (50% w/v) and, after 30 min, filtered through GF 34 filters (Schleicher-Schull), which binds proteins. After washing the filters three times with 50% TCA, they were dried at 85 °C. Radioactivity of the filters was determined in a liquid scintillation counter.19... 

Jandl, J. H., J. K. Inman, R. L. Simmons, and D. W. Allen Transfer of iron from serum-iron-binding protein to human reticulocytes. J. Clin. Invest. 38, 161 (1959). 

Gaur, D., Singh, S., Singh, S., Jiang, L., Diouf, A., and Miller, L. H. (2007). Recombinant Plasmodium falciparum reticulocyte homology protein 4 binds to erythrocytes and blocks invasion. Proc. Natl. Acad. Sci. USA 104,17789-17794. 

Some proteins, such as the poly(A)-binding protein (p78), are present in most if not all mRNPs, whereas others appear to be cell specific and mRNA selective. In unfertilized sea urchin eggs and Xenopus oocytes, for example, untranslated messenger is sequestered by association with proteins that prevent translation until later stages of development. Duck reticulocytes contain globin mRNP, which cannot be translated in vitro, whereas the mRNA obtained by deproteinizing the complex can be translated, showing that in this case translation is prevented by the mRNP proteins. 

The initial identification of a cap-binding protein resulted from experiments by Sonenberg and Shatkin (1977), who developed a crosslinking assay to detect polypeptides which were in physical proximity to the 5 ends of capped mRNAs. Reovirus mRNA was synthesized containing H-label in the methyl groups of the 5 cap. The mRNA was oxidized with NaI04 so as to convert the 2, 3 -cw-diol of the 5 -terminal m G to a reactive dialdehyde, and the oxidized mRNA was then incubated with ribosomal salt wash from rabbit reticulocytes or from Ehrlich ascites cells (Sonenberg et al., 1978). The... 

Lee et al. (1983) attempted to directly measure the effect of mRNA secondary structure on its reaction with cap-binding proteins. The 50,000- and 80,000-dalton, ATP/Mg -dependent CBPs in crude rabbit reticulocyte ribosomal salt wash showed the usual ATP-de-pendence for crosslinking to the oxidized cap structure of native reovirus mRNA. However, when inosine-substituted mRNA was used, specific crosslinking of these polypeptides occurred in the absence of ATP/Mg ". These results were contradicted in a subsequent report (Tahara et al., 1983) which demonstrated that ATP was required for crosslinking purified eIF-4A and 4B to oxidized, inosine-substituted mRNA as well as for authentic ribosome binding by the denatured RNA. The reason for the discrepant results by these two laboratories has not yet been elucidated. Perhaps small amounts of ATP were... 

Transport. Transcobalamin II dehvers the absorbed vitamin 3 2 to cells and is the primary plasma vitamin B22-binding transport protein. It is found in plasma, spinal fluid, semen, and extracellular fluid. Many cells, including the bone marrow, reticulocytes, and the placenta, contain surface receptor sites for the transcobalamin II—cobalamin complex. 

Fig. 1.56. Control of eIF-2 by phosphorylation. Phosphorylated eIF-2 GDP binds strongly to eIF-2B without nucleotide exchange occurring. Initiation of protein biosynthesis is not possible in this case.In reticulocytes, eIF-2 is subject to phosphorylation by the heme-regulated eIF-2-kinase (HRI). The activity of the dimeric HRI is regulated via the heme concentration. Another protein kinase that can phosphorylate and regulate eIF-2 is the RNA-dependent eIF2a-kinase (PKR). The latter is induced by interferons and activated by double stranded RNA.

In a total volume of 300 pi, 50pl reticulocyte membranes in phosphate buffer (500 pg protein) were incubated for 60 min at 25°C with 50 pi (30 nCi) of (-)-3H-CGP-12177, 180 pi of plasma and 20pl of 310 mOsm sodium phosphate buffer (pH 7.4). Standard curves were run for each rabbit separately in order to avoid errors due to possible differences in protein binding. To generate the standard curves, blank plasma was used and incubated in the presence of 1-20 nM timolol. For the determination of non-specific binding, incubation in a 10-5 M propranolol solution was used. 

Schade reviewed (114) the earlier studies on the role of serum transferrin in iron transport. Various early investigators had observed that the blood serum transferrin rapidly bound iron administered either through the gastrointestinal tract or by intravenous injection. There was a rapid turnover of iron in the blood serum and the degree of saturation of the transferrin was related to the amount of iron administered. In no instances, however, was the blood serum transferrin ever saturated with iron. Jandl et al. (71) have shown that both ovotransferrin and serum transferrin can transport plasma iron into red cells and that the transport is dependent on the concentration of transferrin. Iron taken up by the blood cells could not be eluted by subsequent incubation with iron-free transferrin solutions. More recently Morgan and Laurel (99) reported that iron uptake in reticulocytes is independent of the transferrin concentration. The iron complex of serum transferrin has a higher affinity for immature red cells than does the iron-free protein (72). Both bind specifically to immature red cells and the attachment permits the cells to remove the iron. Once the iron is removed, however, the iron-free transferrin can be replaced by an iron-transferrin complex. 

A low molecular weight protein, different from metallothionine which reversibly binds iron with high affinity has been isolated from rabbit reticulocyte cytosol (54, 55, 56). Although very little is yet known about its physiological properties, the molecular weight is around 6000, and iron appears to be reversibly bound under physiological conditions. This protein may be able to mobilize iron from the plasma membrane and donate it for heme and ferritin biosynthesis (56), but no definitive physiological role for siderochelin has been established. 

The dramatic role of the anion can perhaps best be appreciated from simple quantitative considerations. In the absence of a suitable anion, specific binding of iron to transferrin does not occur at all the effective binding constant is zero. At physiologic pH and bicarbonate concentrations, however, the effective binding constant is about 5 X 1023 M"1 24, 50). This means that in 1 L of blood plasma, in which the transferrin is only about 30% saturated with iron, there will be less than one free ferric ion or that a molecule of the ferric—transferrin complex will spontaneously dissociate only about once in 10,000 years. Since iron is readily removed from the transferrin molecule during its interaction with the reticulocyte without disrupting protein structure 51, 52), a... 

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