Biomedical subjects

R E Hileman

Publications and source records attributed to R E Hileman.

At least 19 recordsLinked to original sources

PubMed1997

Interaction of fibroblast growth factor-1 and related peptides with heparan sulfate and its oligosaccharides.

Fibroblast growth factors (FGFs) are a family of angiogenic and mitogenic proteins that promote cell division. The binding of FGFs to the heparan sulfate of cell-surface-bound proteoglycans appears to be critical for their activity. The interaction of fibroblast growth factor-1 (FGF-1 or aFGF) using heparin lyase-derived oligosaccharides from heparan sulfate was investigated. FGF-1 was also shown to protect sequences in heparan sulfate from heparin lyase digestion and protected oligosaccharide products of octasaccharide and decasaccharide size were recovered by FGF-1 affinity chromatography, suggesting that the high-affinity binding of heparan sulfate to FGF-1 resides within an octasaccharide sequence. The FGF-1 binding affinity of heparan sulfate is reduced compared to heparin presumably due to the absence of 6-sulfate groups in heparan sulfate. Inspection of the FGF-1 heparan sulfate binding domain shows that the majority of interacting amino acids are contained within a 20-amino-acid sequence that folds back upon itself (because of three turns) forming a triangular shaped cup of positive charge. The importance of FGF-1 binding site topology was investigated using three synthetic peptide mimics of the FGF-1 glycosaminoglycan (GAG) binding site. Heparan sulfate affinity chromatography and isothermal titration calorimetry, used to measure binding thermodynamics, demonstrated that a synthetic peptide analogous to the GAG binding site in FGF-1 bound tightly to heparan sulfate. A peptide containing a D-proline in place of L-proline bound with considerably reduced affinity, presumably due to the altered structure of the second turn in the binding site. A cyclic peptide, expected to be topologically most similar to the triangular GAG binding site in FGF-1, bound with the highest affinity to heparan sulfate. These data suggest the triangular topology of the GAG binding site in FGF is critical for its interaction with heparan sulfate. Analysis of known GAG binding sites in 25 proteins using the Chou-Fasman algorithm show that these sites commonly contain turns.

Amino Acid Sequence↗

PubMed1997

Pattern and spacing of basic amino acids in heparin binding sites.

Glycosaminoglycan (GAG)-protein interactions regulate a myriad of physiologic and pathologic processes, yet an understanding of how these molecules interact is lacking. The role of the pattern and spacing of basic amino acids (arginine (R) and lysine (K)) in heparin binding sites was investigated using peptide analogs as well as by examining known heparin binding sites. Peptides having the general structure R(n)W (n = 3-9, where tyrosine (W) was added for peptide detection) were synthesized and their interaction with heparin was determined by isothermal titration calorimetry. Binding affinity increased with increasing number of R residues. A 9-mer of R (R9W) bound as tightly to heparin as acidic fibroblast growth factor under physiologic conditions. Despite their high affinity for heparin, long stretches of basic amino acids are uncommon in heparin binding proteins. Known heparin binding sites most commonly contain single isolated basic amino acids separated by one nonbasic amino acid. Peptides having the structure, H3CCONH-GRRG(m)RRG(5-m)-CONH2 (denoted as the RRG(m)RR peptide series) and H3CCONH-GRRRG(m)RG(5-m)-CONH2 (denoted as the RRRG(m)R peptide series), where m = 0-5, were synthesized to test the hypothesis that the spacing of basic amino acids in heparin binding sites is optimally arranged to interact with different GAGs. The peptides, in both the -RRG(m)RR- and -RRRG(m)R- peptide series, when m = 0, bound most tightly with heparin, as measured by affinity chromatography. In contrast, the -RRG(m)RR-peptide series interacted most tightly with heparan sulfate when m = 0 or 1, whereas the -RRRG(m)R- peptide series bound tightest when m = 3. These results are consistent with our understanding of heparin and heparan sulfate structure. A highly sulfated GAG, such as heparin, interacts most tightly with peptides (or peptide sequences within proteins) containing a complementary binding site of high positive charge density. Heparan sulfate, having fewer and more highly spaced negatively charged groups, interacts most tightly with a complementary site on a peptide (or peptide sequences with proteins) that has more widely spaced cationic residues.

Amino Acid Sequence↗

PubMed1997

Glycosaminoglycans can influence fibroblast growth factor-2 mitogenicity without significant growth factor binding.

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.

Animals↗

PubMed1997

Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species.

This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.

Amino Sugars↗

PubMed1997

Polysulfated carbohydrates analyzed as ion-paired complexes with basic peptides and proteins using electrospray negative ionization mass spectrometry.

Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.

Carbohydrates↗

PubMed1997

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

Amino Acid Sequence↗

PubMed1997

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides.

Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.

Animals↗

PubMed1997

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor.

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Animals↗

PubMed1996

Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites.

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.

Animals↗

PubMed1996

Heparin structure and interactions with basic fibroblast growth factor.

Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.

Binding Sites↗

PubMed1995

Differences in the interaction of heparin with arginine and lysine and the importance of these basic amino acids in the binding of heparin to acidic fibroblast growth factor.

Although the interaction of proteins with glycosaminoglycans (GAGs) such as heparin are of great importance, the general structural requirements for protein- or peptide-GAG interaction have not been well characterized. Electrostatic interactions between sulfate and carboxylate groups on the GAG and basic residues in the protein or peptide dominate the interaction, but the thermodynamics of these electrostatic interactions have not been studied. Arginine residues occur frequently in the known heparin binding sites of proteins. Arginine is also more common than lysine in randomly synthesized 7-mer peptides that bind to immobilized heparin and heparan sulfate. We have used heparin affinity chromatography, equilibrium dialysis, and isothermal titration calorimetry techniques to further investigate these interactions. A 7-mer of arginine eluted from a heparin-affinity column at 0.82 M NaCl, whereas the analogous 7-mer of lysine eluted at 0.64 M. Similarly, the putative heparin binding site peptide (amino acid residues 110-130) from acidic fibroblast growth factor, which contained four lysine and two arginine residues, eluted at 0.50 M, whereas the analogous peptide with six lysine residues eluted at 0.41 M and one with six arginine residues eluted at 0.54 M. At 25 degrees C in 10 mM sodium phosphate, pH 7.4, carboxy and amino termini blocked arginine (blocked arginine) bound to heparin twice as tightly as blocked lysine as measured by equilibrium dialysis Similarly, at 30 degrees C in 10 mM sodium phosphate, pH 7.4, and in water, blocked arginine bound 2.5 times more tightly to anions in heparin than blocked lysine. Using titration calorimetry, the enthalpy of blocked arginine and lysine binding to heparin was 1.14 +/- 0.24 and 0.45 +/- 0.35 kJ/mol, respectively, under identical conditions. Our observations show that blocked arginine- and arginine-containing peptides bound more tightly to GAGs than the analogous lysine species and suggest that the difference was due to the intrinsic properties of the arginine and lysine side chains. The greater affinity of the guanidino cation for sulfate in GAGs is probably due to stronger hydrogen bonding and a more exothermic electrostatic interaction. This can be rationalized by soft acid, soft base concepts.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin.

A method for the preparation of heparan sulfate from peptidoglycan heparin is described. The objective of this research was to provide a basis for the development and validation of an industrial process to support the preclinical development of heparan sulfate and/or heparan sulfate derivatives. In the preparation of heparan sulfate, heparin was recovered by alcohol fractionation and dermatan sulfate was isolated by selective precipitation. The remaining crude heparan sulfate was fractionated by anion-exchange chromatography into five subfractions. The biological activities of these subfractions were examined by anticoagulant and amidolytic assays. Molecular weight and molecular size were determined using capillary viscometry and polyacrylamide gel electrophoresis. Charge density and degree of sulfation were determined by cellulose acetate electrophoresis and elemental analysis. Oligosaccharide and disaccharide analysis relied on enzymatic depolymerization using heparin lyases followed by polyacrylamide gel and capillary electrophoresis. 1H NMR analysis provided detailed structural information on each subfraction. Crude heparin sulfate and its subfractions showed significant differences in physical, structural and biological properties.

Animals↗

PubMed1995

Dermatan sulfate as a potential therapeutic agent.

1. Dermatan sulfate is a linear, sulfated polysaccharide and is a glycosaminoglycan component of several important proteoglycans. This minireview discusses the biosynthesis, structure and biological function of dermatan sulfate proteoglycans. 2. Dermatan sulfate and its derivatives are being investigated as a new class of anticoagulant and antithrombotic agents. 3. The preparation, chemistry and structure-activity relationship of dermatan sulfate is described. 4. Dermatan sulfate, low molecular weight dermatan sulfate and glycosaminoglycan mixtures containing dermatan sulfate have been used clinically. 5. The future prospects of these agents and other new, potentially useful dermatan sulfate based therapeutics are discussed.

Animals↗

PubMed1994

Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.

The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.

Animals↗

PubMed1994

Mechanism of action of an eukaryotic initiation factor-2 (eIF-2) associated 67 kDa glycoprotein (p67) and an eIF-2 kinase (dsI).

Mechanism of regulation of eIF-2 alpha-subunit phosphorylation by dsI and p67 was studied. The results are as follows: (1) At low dsI concentration, p67 protected equimolar concentration of eIF-2. (2) At high dsI concentration, dsI efficiently phosphorylated eIF-2 alpha-subunit even when equimolar concentrations of both p67 and eIF-2 were present. Significantly increased p67 concentration was necessary to protect eIF-2 alpha-subunit at high dsI concentration. (3) dsI was also phosphorylated as it phosphorylated eIF-2 alpha-subunit. p67 inhibited both eIF-2 alpha-subunit and dsI phosphorylation similarly. (4) Although the [32P]-labelled dsI formed during the reaction could be effectively chased upon subsequent addition of excess unlabelled eIF-2 and ATP, the [32P] labelled eIF-2 formed under identical conditions, retained most of the radioactivity. (5) dsI coimmunoprecipitated with three subunit eIF-2 and p67 inhibited this coimmunoprecipitation reaction. It has been proposed: Three subunit eIF-2 and free p67 are in equilibrium with eIF-2 bound to p67 and, eIF-2.p67 complex is resistant to dsI phosphorylation. Activated dsI is already phosphorylated. At high concentration, dsI(P) can bind to free three subunit eIF-2 and form eIF-2.dsI(P) complex. dsI(P) in this complex then transfers its phosphoryl residue to eIF-2 and forms eIF-2 alpha(P) in an irreversible reaction. In a subsequent reaction, unphosphorylated dsI is autophosphorylated using [gamma 32P]-ATP and the cycle continues. Inhibition of eIF-2 alpha-subunit phosphorylation by p67 blocks this phosphorylation cycle and consequent dsI phosphorylation.

Aminopeptidases↗

PubMed1993

Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor 2-associated 67-kDa protein (p67).

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit.

Amino Acid Sequence↗

PubMed1993

Characteristics of the eukaryotic initiation factor 2 associated 67-kDa polypeptide.

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p67) protects the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation, and this promotes protein synthesis in the presence of active eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328]. This report presents the results of studies related to characteristics of p67 action and the mechanism of p67:eIF-2 interaction: (1) p67 antibodies inhibited protein synthesis in hemin-supplemented rabbit reticulocyte lysates, and such inhibition was reversed by preincubation of the antibodies, specifically with p67. (2) p67 inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 alpha-subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 beta-subunit. (3) p67 bound specifically to the eIF-2 gamma-subunit. p67 co-immunoprecipitated with the eIF-2 subunits when a p67/eIF-2 mixture was treated with p67 or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubated specifically with the eIF-2 gamma-subunit antibodies, subsequent co-immunoprecipitation of p67 with the eIF-2 subunits was completely inhibited. Similarly, preincubation of p67 and p67 antibodies prevented subsequent p67 binding to eIF-2. Preincubation of eIF-2, with either eIF-2 alpha- or beta-subunit antibodies, had no effect on p67 co-immunoprecipitation with the eIF-2 subunits. (4) p67:eIF-2 interaction is necessary for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Casein Kinases↗

PubMed1988

Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation.

Two protein factors, eIF-2 as well as a high molecular weight protein complex from reticulocyte ribosomal high-salt wash which we term Co-eIF-2, promote Met-tRNA(f) binding to 40S ribosomes. This binding is dependent on the presence of an AUG codon or natural mRNAs [Roy et al. (1984) Biochem. Biophys. Res. Commun. 122, 1418-1425]. Co-eIF-2 contains two component activities, Co-eIF-2A and Co-eIF-2C. Previously, we have purified an 80-kDa polypeptide containing Co-eIF-2A activity and showed that this polypeptide is a component of Co-eIF-2 and is responsible for Co-eIF-2A activity in Co-eIF-2 [Chakravarty et al. (1985) J. Biol. Chem. 260, 6945-6949]. We now report purification of a protein complex (subunits of Mr 180K, 110K, 65K, 63K, 53K, 50K, 43K, and 40K) containing Co-eIF-2C activity and devoid of Co-eIF-2A activity. In SDS-PAGE, the purified Co-eIF-2C preparation and an eIF-3 preparation (purified in Dr. A. Wahba's laboratory) separated into seven similar major polypeptides (Mr 110K, 65K, 63K, 53K, 50K, 43K, and 40K). The 50-kDa polypeptide in Co-eIF-2C was immunoreactive with a monoclonal antibody against eIF-4A (50 kDa). We have studied the roles of purified Co-eIF-2A and Co-eIF-2C activities in ternary and Met-tRNA(f).40S ribosome complex formation. The results are as follows: (1) At low and presumably physiological factor concentration (30 nM), eIF-2 did not form detectable levels of ternary complex. Moreover, such complex formation was totally dependent on the presence of Co-eIF-2A and/or Co-eIF-2C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗