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PubMed1987

Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes.

When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.

Amino Acids↗
PubMed1987

Hypusine formation in eukaryotic initiation factor 4D is not reversed when rates or specificity of protein synthesis is altered.

In mammalian cells, a single major cellular protein (eukaryotic initiation factor 4D) is post-translationally modified by the conversion of a lysine residue into the unusual amino acid hypusine. This modification was reported to occur during mitogen-stimulated growth of lymphocytes but not during quiescence, suggesting that alternative forms of eukaryotic initiation factor 4D might play a role in the regulation of cell growth perhaps through the control of protein synthesis itself (Cooper, H. L., Park, M. H., and Folk, J. E. (1982) Cell 29, 791-797). We took advantage of the drastic changes in translational specificity which occur in heat-shocked cells of Drosophila melanogaster, and of the wide variations in translation rates which occur in response to alterations of growth media in the fungus Saccharomyces cerevisiae, to investigate the relationship between the intracellular level and state of modification of the hypusine-containing protein and the rate and specificity of translation. We also studied whether the hypusine residue in this protein might be subject to further modification or reversion to lysine. Under all conditions examined, the protein was remarkably long-lived. Furthermore, the hypusine persists in this protein as hypusine, without further modification or reversion to lysine. Thus, we observe no correlation between the state of cellular translation and the persistence or reversal of this protein's modification. In addition, the data imply that neither are the state of such key cellular processes as DNA replication, RNA transcription, or carbohydrate metabolism so correlated.

Animals↗
PubMed1988

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells.

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.

Animals↗
PubMed1988

Cell-free synthesis of deoxyhypusine. Separation of protein substrate and enzyme and identification of 1,3-diaminopropane as a product of spermidine cleavage.

The post-translational formation of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) occurs in a precursor of eukaryotic initiation factor 4D by way of two major steps: 1) transfer of the 4-aminobutyl moiety from spermidine to the epsilon-amino group of a specific lysine residue to form an intermediate, deoxyhypusine; 2) hydroxylation of the deoxyhypusine residue to form hypusine. The initial step of this modification, deoxyhypusine synthesis, was studied in fractionated lysates of Chinese hamster ovary cells, untreated, or treated with alpha-difluoromethylornithine (DFMO); the enzyme(s) and the protein substrate (eukaryotic initiation factor 4D precursor) were separated. The enzyme activity was found in the 0-45% ammonium sulfate fraction from both untreated and DFMO-treated cells. The protein substrate was detected in the 45-75% ammonium sulfate fraction from cells depleted of spermidine by treatment with DFMO, but not in any fraction from untreated cells. Upon further purification of the protein substrate by ion exchange chromatography, the requirement for a pyridine nucleotide, notably NAD+, became apparent. Free 1,3-diaminopropane was identified as a spermidine cleavage product formed concurrently with the 4-aminobutyl transfer step of deoxyhypusine synthesis. Compounds structurally related to spermidine, e.g. caldine, N4-benzylspermidine, homospermidine, and a spermine homologue, thermine, as well as 1,7-diaminoheptane, 1,8-diaminooctane, and 1,9-diaminononane caused significant inhibition of deoxyhypusine synthesis presumably due to competition with spermidine. 1,3-Diaminopropane exhibited a potent inhibition of deoxyhypusine formation, probably through a different mechanism.

Animals↗
PubMed1988

Antiviral immunity induced by recombinant nucleoprotein of influenza A virus. I. Characteristics and cross-reactivity of T cell responses.

The gene for the nucleoprotein of the A/NT/60/68 influenza virus was expressed in bacteria and the recombinant protein purified. Lymph node cells from mice immunized with recombinant nucleoprotein proliferated in response to in vitro stimulation with a range of type A influenza viruses. Proliferation was inhibited by mAb to CD4 and class II MHC gene products. IFN-gamma was produced and type-specific CTL were generated in stimulated cultures of immune lymph node cells. These CTL were CD4+ and restricted to class II MHC gene products. Immunization with recombinant nucleoprotein generated Th cells in vivo as measured by the ability to generate an accelerated response to hemagglutinin after challenge with inactivated virus. The results are discussed with reference to a cross-reactive vaccine against influenza.

Animals↗
PubMed1986

Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa).

By a combination of protein chemistry and recombinant DNA methods a glycine-rich region was found to be located near the carboxyl terminus of the nucleolar specific phosphoprotein, nucleolin, from Novikoff hepatoma (protein C23) and Chinese hamster ovary cells (100-kDa nucleolar protein). A sequence of 192 amino acid residues was derived from partial sequences of cyanogen bromide and N-bromosuccinimide fragments of protein C23 and deduced protein sequence from Chinese hamster ovary cell 100-kDa cDNA sequences. The 66 residues sequenced by protein methods were identical to the corresponding residues deduced by DNA sequencing. The multiple residues of NG,NG-dimethylarginine (DMA) contained in the nucleolin polypeptide were found to be limited to a segment of less than 10 kDa near the carboxyl-terminal end of the protein. This segment also contained internally repeated sequences (e.g. 7 copies of the sequence Gly-Gly-Arg-Gly-Gly were found) which were unrelated to sequences closer to the amino-terminal end. Most arginine residues in this region were surrounded by 2 or 3 glycine residues and were relatively close in sequence to phenylalanine residues.

Amino Acid Sequence↗
PubMed1984

[Binding of 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (thrombocyte activating factor) by plasma components. Exchange of the thrombocyte activating factor between lipoproteins and thrombocytes].

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating factor (PAF), to plasma components was studied. Gel filtration and lipoprotein fractionation revealed the presence in the plasma of PAF-binding fractions corresponding to plasma albumin as well as of low and high density lipoproteins. Incubation of PAF-containing lipoproteins with rabbit platelets resulted in a transfer of PAF to the platelets. PAF bound to plasma albumin is less exchangeable than PAF bound to lipoproteins. The PAF-transferring efficiency of high density lipoproteins (HDL) and of low density lipoproteins (LDL) correlates with the amounts of HDL- and LDL-receptors on the platelet surface. It may thus be assumed that PAF released by various cells interacts with lipoproteins which further transport the bound PAF to target cells carrying lipoprotein receptors.

Animals↗
PubMed1984

A hepatocyte receptor for high-density lipoproteins specific for apolipoprotein A-I.

Apolipoprotein (apo) E-deficient rat high-density lipoproteins (HDL) bind to isolated rat hepatocytes at 4 degrees C by a process shown to be saturable and competed for by an excess of unlabeled HDL. Uptake (binding and internalization) at 37 degrees C was also saturable and competed for by an excess of unlabeled HDL. At 37 degrees C the HDL apoprotein was degraded as evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the incubation media. The binding of a constant amount of 125I-apo-E-deficient HDL was measured in the presence of increasing concentrations of various lipoproteins. HDL and dimyristoyl phosphatidylcholine (DMPC) X apo-A-I complexes decreased binding by 80 and 65%, respectively. Human low-density lipoproteins, DMPC X apo-E complexes, and DMPC vesicles alone did not compete for apo-E-deficient HDL binding. However, DMPC X apo-E complexes did compete for the binding of the total HDL fraction that contained apo-E but to a lesser extent than did DMPC X apo-A-I. DMPC X 125I-apo-A-I complexes also bound to hepatocytes, and this binding was competed for by excess HDL (70%) and DMPC X apo-A-I complexes (65%), but there was no competition for binding by DMPC vesicles or DMPC X apo-E complexes. It thus appears that hepatocytes have a specific receptor for HDL and that apo-A-I is the ligand for this receptor.

Animals↗
PubMed1984

Factors controlling the release from human blood polymorphonuclear cells in vitro of a proteolytic activity directed against apolipoprotein A-II.

Human high-density lipoprotein class-3 (HDL3) was incubated with freshly isolated blood polymorphonuclear leukocytes (PMN) at 37 and 4 degrees C. At both temperatures the release of proteolytic activity (PA) causing the specific hydrolysis of apo-A-II was dependent on the concentration of HDL3 in the medium. At 37 degrees C, the efflux of PA was linear and no saturation was reached up to an HDL3 protein concentration in the medium of 800 micrograms/ml. In turn, at 4 degrees C, maximal PA release was reached at a concentration below 600 micrograms/ml of HDL3 protein/ml in the medium. Canine HDL, which contains apo-A-I, but not apo-A-II, was as effective as human HDL3 in promoting the release of PA from PMN. This property was also exhibited by egg lecithin/cholesterol vesicles containing apo-A-I. At 4 degrees C, there was no strict correlation between efflux of PA affected by HDL3 and specific binding of 125I-apo-A-I (HDL3). In competitive binding experiments, a 50-fold excess of unlabeled HDL3 prevented more than 90% of the binding of 125I-apo-A-I (HDL3) to PMN, whereas an excess of unlabeled low-density lipoprotein exhibited no effect. When human HDL3 was incubated with PMN at 4 or 37 degrees C and then subjected to ultracentrifugation at d 1.21 g/ml, most of the PA that was initially associated with this lipoprotein was recovered in the bottom of the tube. By gel filtration, both PA and HDL3 were in the same peak in a low ionic strength buffer, but were dissociated from each other by a high-salt solution (d 1.21 g/ml). We conclude that both naturally occurring HDLs and apo-A-I-stabilized lipid vesicles favor the release from PMN of an enzymatic activity which cleaves human apo-A-II. This release appears to be dependent both on the interaction of the cells with the lipoprotein ligand and on the lipoprotein surface area acting as the acceptor for the enzyme, probably through electrostatic forces.

Animals↗
PubMed1982

Correlation of low and high density lipoprotein binding in vivo with rates of lipoprotein degradation in the rat. A comparison of lipoproteins of rat and human origin.

These studies were done in the rat to correlate the ability of low and high density lipoproteins of rat (rLDL and rHDL) and human (hLDL and hHDL) origin to bind in vivo to specific tissues with the rates at which these same lipoprotein fractions were cleared from the circulation. The adrenal gland and liver manifested the greatest amounts of rLDL binding in vivo, but activity also was found in spleen, lung, kidney, ovary, and intestine. In contrast, little or no such binding was found utilizing either methyl-rLDL or hLDL. rHDL containing E apoprotein bound to the same group of tissues although in lesser amounts, except in the case of ovary and adrenal gland which bound disproportionately greater amounts of rHDL than rLDL. In keeping with these marked differences in tissue binding, the clearance of rLDL from the plasma equaled 847 +/- 36 microliters/h/100 g of rat while that of methyl-rLDL and hLDL was only 368 +/- 8 and 363 +/- 11 microliters/h/100 g of rat, respectively. When the steady state plasma level of rLDL was raised 2.5-fold, the clearance decreased slightly to 705 +/- 20 microliters/h/100 g of rat. The clearance of hLDL remained constant, however, at about 350 microliters/h/100 g of rat even when the plasma hLDL level was raised to very high values. The clearance of rHDL and hHDL equaled 644 +/- 16 and 408 +/- 13 microliters/h/100 g of rat, respectively, reflecting the more similar rate of binding of rHDL and hHDL to the tissues of the rat. Rates of whole animal sterol synthesis were lowered from 28 mumol/h to 8.8 mumol/h or 13 mumol/h by fasting and cholesterol feeding, respectively, and stimulated to 71 mumol/h by cholestyramine treatment. Under these same conditions, hepatic cholesterol synthesis could be lowered from the normal rate of 15 mumol/h to 4.2 mumol/h and raised to 50 mumol/h. None of these treatments, however, affected the plasma clearance of rLDL and rHDL. In contrast, treatment with ethinyl estradiol increased by 3-fold both the hepatic binding and the whole animal plasma clearance of rLDL. Following resection of approximately two-thirds of the liver under carefully controlled metabolic conditions, there was no change in the rate of hepatic cholesterol synthesis or rLDL binding in the remaining liver, but the clearance of chylomicrons, rLDL, and rHDL diminished by 67%, 26%, and 17%, respectively, suggesting that in the rat the liver was responsible for the degradation of approximately 97%, 39%, and 27%, respectively, of these lipoprotein fractions.

Animals↗
PubMed1983

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin.

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.

Animals↗
PubMed1983

Control of lipopolysaccharide-high density lipoprotein binding by acute phase protein(s).

When Salmonella minnesota R595 lipopolysaccharide (LPS) is mixed with serum, the LPS eventually forms a complex with high density lipoprotein (HDL). Complex formation is conveniently followed by CsCl equilibrium density gradient centrifugation. When mixing 10 micrograms LPS with normal rabbit serum (NRS) at 37 degrees C in the presence of 20 mM EDTA, the half-life for LPS binding to HDL is typically 2 to 3 min. When the same experiment is performed with the use of acute phase rabbit serum (APRS; collected 24 hr post-induction with silver nitrate), the half-life for LPS binding to HDL is typically 40 to 100 min. Thus LPS binding to HDL occurs some 20- to 40-fold slower in APRS than in NRS. Two other phenomena have been found, the time dependencies of which correlate well with the time dependency of LPS binding to HDL in APRS. If LPS-APRS reaction mixtures are cooled to 4 degrees C shortly after mixing and are dialyzed against 2.5 mM HEPES, 15 mM NaCl, pH 7.4 buffer, LPS is recovered in the washed precipitates ("euglobulin precipitate") if, and only if, the LPS-HDL binding reaction is not complete. The amount of LPS in the precipitate correlates well with the amount of LPS that has not bound to HDL. The second phenomenon we observe is that the LPS-containing euglobulin precipitate prepared from LPS-acute phase serum reaction mixtures shortly after mixing also contains a protein, gp60, the concentration of which in the euglobulin precipitate correlates well with the amount of LPS in the precipitate. Thus three phenomena are kinetically well correlated in APRS: the degree of binding of LPS to HDL, the degree of appearance of LPS in a euglobulin fraction, and the concentration of protein gp60 in the euglobulin fraction. We were unable to precipitate gp60 from APRS in the absence of LPS, from APRS after the LPS has fully bound to HDL, or from normal serum in the presence or absence of LPS. The known properties of gp60 are not reminiscent of any other known acute phase reactant. These data demonstrate that APRS contains acute phase reactants that interact with LPS to modify its buoyant density, its solubility, and the rate of its binding to HDL.

Acute Disease↗
PubMed1983

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line.

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Initial velocity kinetic analysis of 30 S initiation complex formation in an in vitro translation system derived from Escherichia coli.

The analysis of initial velocity kinetic data was used to examine the order in which fMet-tRNA and the coat cistron of genomic bacteriophage R17 or Q beta RNA bind to the 30 S ribosome subunit. These data were obtained using a quantitative assay for protein synthesis in Escherichia coli extracts where the rate of accumulation of protein product is dependent on the concentration of mRNA and is partially dependent on fMet-tRNA. Under the conditions of this assay, the amount of protein synthesized was proportional to the formation of ternary complexes between the mRNA, fMet-tRNA, and the 30 S ribosomal subunit. The results from the initial velocity and alternative substrate experiments are consistent with a rapid equilibrium ordered mechanism as opposed to a rapid equilibrium random mechanism. Analysis of the rate of coat protein synthesis at varied concentrations of mRNA and fixed concentrations of fMet-tRNA indicated that fMet-tRNA was the first substrate to bind to the 30 S subunit when either coat cistron was used as the mRNA. This scheme assumes the existence of a relatively slow step in protein synthesis that occurs after both the initiating tRNA and mRNA are bound to the ribosome and which allows substrate addition to reach thermodynamic equilibrium.

Capsid↗
PubMed1983

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis.

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.

Animals↗