Membrane from reticulocytes

Native enzyme is inactivated initially without loss of antibody reactivity. The inactivation reaction is catalyzed by a membrane protein present in all liver membranes but at highest specific activity in plasma membranes and at lowest activity in lysosomal membranes. Inactivation is greatly accelerated in the presence of disulfides such as oxidized glutathione or cystine and retarded by thiols. Disulfides on the membrane protein are implicated because treatment of membranes with dithiothreitol in the presence of iodoacetamide destroys the capacity to inactivate phosphoenolpyruvate carboxykinase. This treatment would reduce and fix protein disulfides. Inactivation requires a membrane protein that shows some tissue specificity, since plasma membranes from reticulocytes or erythrocytes are not active, nor are liposomes prepared from the lipids of liver microsomes. 

Plasma membranes from rabbit reticulocytes, but not those from erythrocytes, are able to glycosylate endogenous proteins through dolichyl-bound sugar derivatives. ... 

Timolol concentration in plasma and tear fluid samples was measured using a modified radioreceptor assay [7]. In the assay, displacement of a P-antagonist, (-)-3H-CGP-12177, from 0-receptors of rat reticulocyte membranes by timolol was measured. Rat reticulocyte membranes were obtained as described by Wellstein el al. [7]. 

Within the past few years, there has been considerable progress in understanding the role played by the mitochondria in the cellular homeostasis of iron. Thus, erythroid cells devoid of mitochondria do not accumulate iron (7, 8), and inhibitors of the mitochondrial respiratory chain completely inhibit iron uptake (8) and heme biosynthesis (9) by reticulocytes. Furthermore, the enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) which catalyzes the insertion of Fe(II) into porphyrins, appears to be mainly a mitochondrial enzyme (10,11,12,13, 14) confined to the inner membrane (15, 16, 17). Finally, the importance of mitochondria in the intracellular metabolism of iron is also evident from the fact that in disorders with deranged heme biosynthesis, the mitochondria are heavily loaded with iron (see Mitochondrial Iron Pool, below). It would therefore be expected that mitochondria, of all mammalian cells, should be able to accumulate iron from the cytosol. From the permeability characteristics of the mitochondrial inner membrane (18) a specialized transport system analogous to that of the other multivalent cations (for review, see Ref. 19) may be expected. The relatively slow development of this field of study, however, mainly reflects the difficulties in studying the chemistry of iron. 

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 uptake of iron from transferrin by reticulocytes is a time-, temperature-, and energy-dependent process in which integrity of both protein and cells is required (64, 65). Synthetic iron chelates, once thought to be effective iron donors (66), appear to depend on membrane-bound transferrin as an intermediate agent cells depleted of the protein by preincubation and washing no longer accept iron from such complexes (67). When such cells are reincubated with transferrin, their capacity to accept iron initially bound to synthetic chelators is largely restored. 

The existence of specific receptors for transferrin on the reticulocyte membrane was implied by the work of Jandl and associates, who observed that trypsin virtually abolished the ability of reticulocytes to take up iron from transferrin without affecting other metabolic functions of the cells (8). Whether the effect of the enzyme was to cleave the receptor from the cell membrane or to degrade it proteolytically was not clear. Neuraminidase treatment also depressed iron uptake by reticulocytes, but to a much lesser degree than trypsin and only at much higher concentrations than needed to abolish the hemagglutinating effects of influenza virus. Subsequent work from Morgan s laboratory has confirmed these results and has shown further that binding of transferrin to the receptor protects it from proteolytic enzymes (70). 

Reticulocytes were obtained from rabbits made anemic by repeated bleeding. Reticulocyte counts were generally 20-80% of total red cells. Standard methods were followed for preparing and 125I-labeling of rabbit transferrin and its incubation with reticulocytes (54). Reticulocyte membranes were prepared by the method of Dodge (77) and solubilized for... 

Figure 2. Loss of membrane proteins during reticulocyte maturation. Reticulocyte-poor (A) and reticulocyte-rich (B) erythrocyte membrane ghost proteins were subjected to 2DE and stained with Coomassie blue (0.5%). Selected spots identical between the protein samples are identified with black boxes. Several of the many spots that are unique to reticulocyte-rich erythrocytes are shown with the black circles. (Reproduced with permission from Prenni and Olver Proteomics A review and an illustration. Veterinary Clinical Pathology, in press.)...

V.Z. Lankin, H. Kuhn, C. Hiebsch, T. Schewe, S.M. Rapoport, A.K. Tikhaze and N.T. Gordeeva, On the nature of the stimulation of the lipoxygenase from rabbit reticulocytes by biological membranes, Biomed.Biochim.Acta 44 (1985) 655-664. 

The iron inserted into apo-heme is acquired from the transferrin circulating in the bloodstream. Transferrin is taken up by cells according to their need for iron. This transport is mediated by a membrane-boimd protein of the plasma membrane called the transferrin receptor. The erythropoietic cell contains a large number of transferrin receptors in its plasma membrane. Stem cells contain very few transferrin receptors because of their lack of hemoglobin synthesis. The normoblast contains 0.3 to 0.8 million transferrin receptors, whereas the more mature reticulocyte contains 0.1 million receptors. After release of iron, transferrin returns to the bloodstream. Most of the transferrin contains iron derived from recently catabolized red blood cells, as shown in Figure 10.33. About 99% of the transferrin contains iron acquired from macrophages, where old red blood cells are dismantled only 1% is derived from recently absorbed dietary iron. 

A unique lipoxygenase was isolated and purified from rabbit reticulocytes [212], The purified enzyme with a molecular weight of 78000 contains two moles of iron per enzyme molecule. The enzyme is identical with the previously known protein factors which inhibit the respiratory chain. The purified lipoxygenase causes a loss of acid-labile sulfur from mitochondrial electron transfer particles and acts preferably on the mitochondrial membrane. The enzyme is considered to work in the lysis of mitochondria during the maturation of erythrocytes. The major product from arachidonic acid is 15S-hydroperoxy-5,8,l 1,13-eicosatetraenoic acid, although the 12S-hydroperoxy derivative is produced as a minor product [213]. 

Ribonucleoprotein complexes containing mRNA have been isolated from free polysomes of reticulocytes (Huez et al., 1967 Weisberger and Armentrout, 1966), of rat liver (Henshaw, 1968), from the membrane-bound polysomes of rat liver (Leytin et al., 1970), from thyroid gland (Cartouzou et al., 1968) and from L-cells (Perry and Kelley, 1968). 

A very important question concerns the nature of the proteins of polysomal mRNP. This question has not yet been answered because of the difficulties in the isolation of polysomal mRNPs. Since reticulocyte mRNPs can now be obtained in a pure state, the question may be answered in the near future. In another approach, Schweiger and Hannig (1970) tried to find proteins typical of nuclear D-RNP by direct analysis of polysomes. They separated the total polysomal protein into several fractions using free flow electrophoresis and then compared the fractions with nuclear D-RNPs obtained by means of polyacrylamide gel electrophoresis. In free polysomes and in membrane-bound polysomes, proteins similar to proteins of the nuclear D-RNP were found. If the membrane-bound polysomes are separated from the membranes by treatment with pure deoxycholate, these proteins are dissociated from polysomes and thus resemble the proteins of nuclear D-RNP in their sensitivity to deoxycholate. 

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