Synthesis of non-globin proteins in rabbit-erythroid cells. Synthesis of a lipoxygenase in reticulocytes.

Peripheral rabbit reticulocytes synthesize at least 30 non-globin proteins. One of them is identified as a characteristic lipoxygenase on the basis of its molecular weight, its immunological properties and its behaviour on an ion-exchange column. The enzyme is not produced in bone marrow cells. The synthesis of the lipoxygenase in peripheral blood cells commences on the 3rd day of a bleeding anaemia, increases up to the 5th day and stays constant thereafter at least up to the 14th day. It is concluded that the appearence of the lipoxygenase, which plays a key role in the degradation of mitochondria in the course of maturation of reticulocytes to erythrocytes, is regulated at the translational level.     

Synthesis of delta crystallin from embryonic chick lens messenger ribonucleoprotein complex

A single, major 21 S messenger ribonucleoprotein (mRNP complex) was isolated and purified by sucrose gradient centrifugation after EDTA treatment of high salt washed polysomes from 15 day embryonic chick lenses. A 17 S mRNA was released from the 21 S mRNP. The 21 S mRNP complex coded for a 50 000 molecular weight protein identical to the subunit of delta crystallin. Similar results were obtained with the 17 S mRNA released from the 21 S mRNP complex.     

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.     

Partial purification of the alpha-tubulin and beta-tubulin messenger RNAs from rat brain

Poly(A)-containing RNA from frozen adult rat brain were fractionated by centrifugation in a formamide/sucrose gradient. Individual fractions were used to program protein synthesis in vitro in a reticulocyte lysate. The cell-free translation products were analyzed by two-dimensional electrophoresis in polyacrylamide slab gels. We observed a heterodispersion of the mRNA translation activity coding for the beta-tubulin subunit which contrasts with a relatively homogeneous distribution of the alpha-tubulin subunit mRNA. These last mRNA species are present in a peak which sediments near the 18-S region of the gradient whereas the beta-tubulin mRNA activity is predominant in the fractions corresponding to the heaviest mRNA species. When these heaviest RNAs were separated again by centrifugation in a second formamide/sucrose gradient, a poly(A)-rich RNA population was obtained that was enriched in RNA for programming the beta-tubulin subunit. Analysis of the products whose synthesis in vitro was directed by this mRNA population revealed that beta tubulin was the main protein formed, the ratio beta/alpha being more than tenfold greater than in the products translated in vitro using total poly(A)-rich RNA.     

Messenger ribonucleoprotein particles in silkmoth oogenesis

Silkmoth oocytes contain significant amounts of nontranslating cytoplasmic messenger RNA apparently stored until fertilization. The physical state of this mRNA was examined by bouyant density centrifugation on cesium chloride gradients. Messenger elements were isolated either by oligo(dT) cellulose chromatography or by separation of ooplasm on sucrose gradients. After CsCl density gradient centifugation the mRNA particles banded in a region (1.42–1.48 g/c³) which would indicate a substantial protein content. Electron microscope examination of mRNP fractions revealed particles ranging in size from 180–250 Å.     

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17–20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as old mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of -globin in all systems, while the presence of the cap analogue m⁷G(5)p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5 terminal cap. Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.     

Separation of haemopoietic cells for biochemical investigation. Preparation of erythroid and myeloid cells from human and laboratory-animal bone marrow and the separation of erythroblasts according to their state of maturation.

The separation of haemopoietic bone-marrow cells by centrifugation through discontinuous density gradients of Percoll is described. This method was used to prepare fractions enriched in erythroblasts, myeloid blast cells or reticulocytes from bone marrow of anaemic and non-anaemic rabbits, from the marrow of other anaemic laboratory animals and from human samples. It is a simple, rapid, reproducible and inexpensive technique that can be readily adapted to suit individual requirements. Secondly, a convenient method is presented for the separation of large quantities of bone-marrow cells into fractions enriched in erythroblasts at different stages of maturation, by velocity sedimentation through a linear gradient of 1-2% sucrose at unit gravity. In vitro, erythroblasts adhere together strongly via a mechanism almost certainly involving a beta-galactoside-specific surface lectin termed erythroid developmental agglutinin. Since the efficiency of cell-separation techniques depends heavily on the maintenance of a single cell suspension in which each unit can move independently, the presence of an adhesive molecule at the cell surface is of considerable significance. The effect of washing the marrow with a lactose-containing medium, which has been shown to remove the agglutinin, was therefore investigated in relation to both methods. The separation on Percoll gradients is considerably enhanced by this treatment. In addition, the unit-gravity sedimentation gradient can be loaded with 5-10 times more cells after lactose extraction in comparison with intact marrow. Although enrichment is less, a useful fractionation according to maturation is still obtained.     

Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell- free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Heterogeneity of globin protein synthesis in bone marrow cells of patients with homozygous beta-thalassemia from Tadzhikistan

The synthesis of globin proteins in blood reticulocytes of homozygous beta-thalassemic patients from Tadzhikistan has been previously studied. beta-thalassemia with sharp repression of beta-globin protein synthesis (alpha/beta greater than 10) has been shown to be most representative for the region. In this work, the synthesis of globin proteins has been studied in bone marrow cells of homozygous beta-thalassemic patients. Comparison of data on globin synthesis in bone marrow cells and in blood reticulocytes of the patients has revealed that in some cases the disbalance of chain synthesis in both cell types is equal. In other cases the disbalance in bone marrow cells is less than in blood cells, indicating the instability of beta-globin mRNA that is partially degrading in the process of cell maturation. Homozygous beta-thalassemic cases with low content of Hb F in blood cells (5-10%), with substantial disbalance of alpha and beta-globin synthesis and marked production of gamma-globins in bone marrow cells and in blood reticulocytes are of special interest. It has been assumed that parallel to beta-thalassemia some instability of gamma-globin proteins takes place in these patients.     

Proteomic characterization of messenger ribonucleoprotein complexes bound to nontranslated or translated poly(A) mRNAs in the rat cerebral cortex

Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.     

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos

The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation. The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II. Among the exogenous proteins initiation factor eIF2 is the best substrate and is 7.8 times more efficiently phosphorylated than casein. Endogenous mRNP protein substrates have a Mr of 125000, 65000, 38000, 26000 and 23500. The main phosphate acceptor is the Mr38000 poly(A)-binding protein. Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property. The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed.Abbreviations PMSF phenylmethanesulfonyl fluoride - mRNP messenger ribonucleoprotein - iRNA small inhibitor RNA - eIF2 eukaryotic initiation factor 2     

Initiation factor eIF2 associated with non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina

Non-polysomal poly(A)-containing mRNP of A. salina cryptobiotic embryos is separated in mRNP active in protein synthesis and in repressed mRNP by sucrose gradient centrifugation. In the translationally active fraction the presence of eukaryotic initiation factor 2 (eIF2) is demonstrated by electroblotting of sodium dodecylsulphate/polyacrylamide gels on nitrocellulose and anti-eIF2 antibody detection. mRNP proteins with Mr of 40 000 and 42 000 are identified as the alpha and beta subunits of eIF2. The repressed mRNP is devoid of eIF2 and is associated with an inhibitor ribonucleoprotein composed of a small 85 +/- 2-nucleotide-long RNA and a protein with Mr of 64 000. The latter ribonucleoprotein is a potent inhibitor of the translationally active mRNP.     

Intracellular distribution of ribonuclease activity during erythroid cell development.

Five ribonuclease activities, separable by polyacrylamide gel electrophoresis, have been detected in erythroid bone marrow cells from anaemic rabbits. Their intracellular distribution has been investigated and compared with that of the ribonucleases in reticulocytes. Both the acid and alkaline ribonuclease activities of reticulocytes are much lower (30--50 fold) than those of bone marrow erythroid cells. The most marked decrease in enzyme activity occurs in the fractions containing ribosomes and mitochondria plus lysosomes. In these subcellular organelles there was also a qualitative change in the ribonuclease electrophoretic pattern, whereas the cytosol enzymes of marrow erythroid cells and reticulocytes remained largely unchanged. Several ribonucleases released from reticulocyte membranes with urea were similar to those present in the lysosomal plus mitochondrial fraction, as shown by detection of enzyme activity after polyacrylamide gel electrophoresis. The decline in ribonuclease activity was found to begin in the orthochromatic cells, which have a highly condensed nucleus and are no longer active in DNA and RNA synthesis, and to coincide with a decrease in acid phosphatase activity and loss of lysosomes.     

The lipoxygenase of reticulocytes. Purification, characterization and biological dynamics of the lipoxygenase; its identity with the respiratory inhibitors of the reticulocyte.

A lipoxygenase has been purified from rabbit reticulocyte-rich anaemic blood cells. It possesses a molecular weight of 78 000 and an isoelectric point of 5.5 and contains 5% neutral sugars and two iron atoms per enzyme molecule. The lipoxygenase has proved to be identical with the inhibitors of respiratory proteins described formerly. The actions of the lipoxygenase on linoleic acid, phospholipids, mitochondrial and erythrocyte membranes and electron transfer particles were studied. A special feature of the reticulocyte lipoxygenase is the suicidal character of its action on lipids. With electron transfer particles the reticulocyte lipoxygenase causes a loss of acid-labile sulfur which accompanies respiratory inhibition; the strong respiratory inhibition is not exerted by soybean lipoxygenase. The reticulocyte lipoxygenase acts preferably on mitochondrial membranes as compared with cell membranes of the erythrocyte; erythrocyte cytosol moderates the action on mitochondrial membranes. Furthermore, the lipoxygenase reaction can concomitantly and irreversibly inactivate sulfhydryl enzymes as demonstrated with muscle glyceraldehyde-3-phosphate dehydrogenase. The occurrence of the lipoxygenase here described is restricted to reticulocytes; very low amounts were observed in bone marrow and no lipoxygenase was detectable in normal blood. During the course of an experimental anaemia the lipoxygenase is produced owing to superinduction in large amounts, which may persist for a long time since they escape inactivation. Preliminary evidence was obtained for the occurrence of other lipoxygenases in tissues of lung, spleen, kidney and also epithelial tumours.     

Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes

Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous [1-14C]-arachidonic acid to 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE). Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase. Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is also present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrence of an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradation of mitochondria.