Studies on the mechanism of interaction between rabbit transferrin and reticulocytes

The two stages in the uptake of transferrin by rabbit reticulo-cytes were investigated using radioiodine-labeled rabbit transferrin and albumin. The first stage of rapid, temperature-insensitive uptake of transferrin was similar to albumin uptake: uptake of both proteins increased linearly with increasing protein concentration of the incubation medium up to at least 60 mg/ml, was maximal at low ionic strength and pH, and increased in the presence of basic polyamino acids. Transferrin uptake was in part dependent on the reticulocyte concentration of the blood, but albumin uptake was independent of reticulocyte concentration. The second slower, temperature-sensitive stage of transferrin uptake was linearly related to reticulocyte concentration, and was not found with albumin, α¹-macroglobulin or γ-globulin. Transferrin uptake was optimal at physiological pH and ionic strength and was unaffected by basic polyamino acids. When the transferrin concentration was raised, uptake increased to reach a maximum at a concentration of 15 mg/ml. It was concluded that the first stage of transferrin uptake was in part or wholly due to non-specific adsorption of transferrin to erythrocytes, while the second stage of uptake was specific for transferrin and reticulocytes and depended upon normal function of the cells.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

Changes in surface-membrane components during the differentation of rabbit erythroid cells.

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

The anion-transfer system of erythrocyte membranes. N-(7-Nitrobenzofurazan-4-yl)taurine, a fluorescent substrate-analogue of the system.

The fluorescent probe Nbd-Tau [N-(7-nitrobenzofurazan-4-yl)taurine] was synthesized and evaluated as a potential substrate of the anion-transport system of human erythrocyte membrane. The probe inhibited Cl- exchange in a competitive manner from either surface of the membrane, displaying Ki values in the mM range at the inner surface and in the microM range at the outer surface. Inhibition from within cells was via interaction with Cl--transport sites, whereas from it was via interaction with sites of unidentified nature. Nbd-Tau efflux from cells was monitored fluorimetrically in a continuous mode by a novel method that circumvents separation of the cells from the medium. Using this method, it is shown that Nbd-Tau efflux fulfils the following criteria of a substrate of the anion transport system: (a) susceptibility to classical and specific inhibitors of the system; (b) competitive inhibition with Cl- for anion-transport sites; and (c) temperature coefficient comparable with that of Cl- exchange. The fluorometric method is highly sensitive, versatile, and kinetically informative. With minor modifications it can be used for measuring anion transport across "ghost" and isolated membrane vesicles.     

Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.     

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intra-cellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.     

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.     

Chromatographic separation of the isoforms of the ribosome inactivating protein, gelonin.

The three isoforms of gelonin were separated by affinity chromatography on concanavalin-A Sepharose into discrete components of Mr 31,500, 30,000 and 29,200. Their separation was achieved by apparent differences in interaction with the lectin due to variation in carbohydrate patterns. The Mr 30,000 component representing 67% of the total mixture was the most active in inhibiting protein synthesis in a cell free translation assay using rabbit reticulocyte lysates, although the other two were also active. An antibody prepared against the major fraction (Mr 30,000) reacted well with all three components, demonstrating immunological similarity. This purification may aid the structural elucidation of gelonin and preparation of hormonotoxins and immunotoxins.     

Photosynthetic Prokaryotes. Cell Differentiation and Functions : Edited by G.C. Papageorgiou and L. Packer Elsevier Biomedical; New York, 1983 405 pages. $65.00

A protein (designated as luffin) with an app. M_r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate (IC₅₀, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10⁶ to 1/10⁵ that of ricin.     

Use of an antibody to characterize and determine the role of the major Met-tRNAf deacylase from rabbit reticulocyte ribosomes

Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(alpha) results, secondarily, in the enzymatic deacylation of Met-tRNAf on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNAf deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNAf deacylase II and has no effect on Met-tRNAf deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNAf deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 microgram/ml poly(I X C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNAf and increase the level of Met-tRNAf bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNAf in the 48 S complexes that accumulate.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

Inactivation of eucaryotic ribosomes by the toxic plant proteins abrin and ricin

The effect of abrin and ricin on protein synthesizing systems from different sources was studied. The protein synthesis in a cell-free system from rabbit reticulocytes and from rat liver was strongly inhibited by the toxins, whereas a system from E. coli was not affected. Separate treatments of ribosomes and postribosomal supernatant from a rabbit reticulocyte lysate showed that the site of action of the toxins is on the ribosomes. The inactivation of the rabbit reticulocyte lysate by the toxins was a function of the incubation time and temperature. Protein synthesis was not necessary for the toxins to exert their effect. The data indicate that abrin and ricin act only on the eucaryotic type of ribosomes, and that they exert their effect by enzymatic action.     

Partial purification and characterization of heat stable protein phosphatase inhibitor-2 from rabbit reticulocytes

Previous studies indicated that the species of type 1 and type 2 protein phosphatases (PP-1, PP-2) in rabbit reticulocytes are similar to those of rabbit skeletal muscle and rabbit liver. Reticulocyte PP-1 was found to be selectively inhibited by the heat stable protein phosphatase inhibitor-2 (I-2) from rabbit skeletal muscle. Of interest was the observation that muscle I-2 appeared to regulate protein synthesis in reticulocyte lysates by inhibiting an eIF-2 alpha phosphatase with type 1 properties. In this study we have characterized reticulocyte inhibitor-2 (I-2) and find that its properties are similar to those of skeletal muscle I-2. (i) Both I-2 species are stable to boiling and to acid treatment, and have similar chromatographic profiles on DEAE-cellulose and on Blue Sepharose CL-6B. (ii) The two I-2 species migrate electrophoretically as 26-28,000 dalton polypeptides in SDS-acrylamide gels. (iii) Both skeletal muscle I-2 and reticulocyte I-2 selectively inhibit isolated reticulocyte PP-1 and endogenous PP-1 in the lysate. (iv) Reticulocyte I-2 co-chromatographs with PP-1 on DEAE-cellulose, and over 90% of lysate I-2 can be isolated from this partially purified PP-1. (v) Both inhibitor-2 species are active in the unphosphorylated state, but upon addition to lysates, both are phosphorylated by endogenous cAMP-independent protein kinase(s). In addition a preliminary analysis using a polyclonal antibody against muscle inhibitor-1 confirmed biochemical analyses which indicate that lysates are deficient in inhibitor-1.     

Role of cytoplasmic mRNP proteins in translation.

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per mol of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA approximately 2:1, mol/mol), this protein promotes the translation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte membrane proteins. Sequential accumulation in the membrane during reticulocyte maturation

Reticulocytes of increasing maturity were separated by dextran gradient centrifugation. The accumulation in the membrane of the anion transport protein and other erythrocyte membrane proteins was studied during reticulocyte maturation by separating reticulocytes after incubation with [³⁵S]methionine. The incorporation of the reticulocyte membrane proteins was shown to be sequential, the anion transport protein being inserted at a very early stage in the cells' maturation.     

In vitro biosynthesis of the human cell surface receptor for transferrin

The human cell surface receptor for transferrin is a transmembrane phosphoglycoprotein composed of two disulphide linked and apparently identical subunits of Mr 90 000. Using an affinity purified, polyclonal rabbit antibody, we have studied the in vitro biosynthesis of this receptor. The primary translation product, synthesised in a rabbit reticulocyte lysate programmed with human placental RNA, appears to have the same Mr (78 000) as the unglycosylated molecule immunoprecipitated from tunicamycin-treated cells. In the presence of a dog pancreatic microsomal system the cell free system accurately reproduces the glycosylation and the asymmetric transmembrane integration.     

Limitation of reticulocyte transfer RNA in the translation of heterologous messenger RNAs.

The effect of various tRNAs on protein synthesis was investigated using a tRNA-dependent cell-free system from Ehrlich ascites cells. Ascites cell tRNA and rabbit liver tRNA were found to promote efficient translation of globin mRNA, oviduct mRNA, and encephalomycarditis (EMC) viral RNA. In contrast, reticulocyte tRNA participated efficiently only in the translation of globin mRNA; the translation of oviduct mRNA AND EMC viral RNA in the presence of reticulocyte tRNA resulted in the synthesis of relatively few large mature proteins and the accumulation of discrete, smaller polypeptides. These results suggest that isoaccepting tRNA species required for the synthesis of ovalbumin and EMC viral protein (but not hemoglobin) are probably functionally absent in reticulocyte tRNA, causing a premature, nonrandom termination of synthesis of these proteins. This provides preliminary evidence that variations in tRNA populations, frequently observed between different cell types, are large enough to define and perhaps regulate the proteins that the cell is capable of synthesizing.