Shape and location of eukaryotic initiation factor eIF-2 on the 40S ribosomal subunit of rat liver. Immunoelectron-microscopic and hydrodynamic investigations

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.     

Structure and location of initiation factor eIF-3 within native small ribosomal subunits from eukaryotes

Native small ribosomal subunits (40SN) from rat liver and rabbit reticulocytes prepared at different KC1 concentrations have been investigated by electron microscopy after negative staining. Subunits of both origins show identical features. The initiation factor eIF-3 is located in the middle region of the convex rear side of the particles and covers an area extending from the protuberance at the interface up to the external surface. eIF-3 has the shape of a flat triangular prism and is attached with its triangular base to the ribosomal surface.     

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.     

Physical properties of the purified cardiac muscarinic acetylcholine receptor

The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000.     

Supramolecular structure of the recombinant murine small heat shock protein hsp25

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere-like shape and diameters of 15-18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.     

Physicochemical and kinetic properties of acid phosphatase from Saccharomyces cerevisiae

Acid phosphatase from yeast Saccharomyces cerevisiae was purified, and its physicochemical and kinetic properties were investigated. The sedimentation coefficient has been determined to be s0(20,w) = 13.6 S. The diffusion constant has been found to be 3.9 X 10(-7) cm2s-1, and the calculated partial specific volume was v = 0.663 cm3/g. From these data, a molecular weight of 252,000 was calculated. Electrophoresis on gel slabs, with a linear concentration gradient of polyacrylamide (4-30%), showed size heterogeneity of the native enzyme preparation and indicated an apparent molecular weight in the range of 170,000 to 360,000. In the presence of sodium dodecyl sulfate, the molecular weight was in the range of 82,000 to 165,000, indicating dimeric structure of the native enzyme, which was confirmed by cross-linking experiments. Isoelectric focusing demonstrated charge heterogeneity of enzyme preparation. From CD spectrum it was calculated that the enzyme contains about 29% of alpha-helical structure. Excitation at 278 nm gave an emission fluorescence spectrum with a maximum at 340 nm. Amino acid analysis revealed a high content of aspartic acid, serine, and threonine. Glycine is found as the NH2-terminal amino acid. Initial velocity dependence on substrate concentration, as well as on pH, and thermostability studies indicated the presence of at least two enzyme forms in the preparation.     

Size and shape of bovine interphotoreceptor retinoid-binding protein by electron microscopy and hydrodynamic analysis

Individual molecules of interphotoreceptor retinoid-binding protein (IRBP), a protein likely to be important in the visual cycle, were visualized by means of electron microscopy. IRBP was coated with a very thin layer of tungsten and photographed by dark-field imaging. IRBP is seen to be a flexible, elongated molecule about 24 nm in length by 3-4 nm in width (statistical modes). These dimensions agree very well with those calculated from the frictional ratio obtained from sedimentation data. Approximately half of these rod-shaped IRBP molecules are straight, and half are bent in the middle, usually with an angle of 60-90 degrees between the two arms. A representation of IRBP as a bendable string of beads yields calculations of dimensions and of hydrodynamic parameters consistent with the electron microscopic and sedimentation data; the sedimentation coefficients derived from this representation are nearly insensitive to molecular bending. When IRBP is bound to saturating amounts of its endogenous ligands, all-trans- or 11-cis-retinol, its sedimentation behavior is unchanged, and the same types of particles are visualized by electron microscopy as with the free protein; however, a greater proportion of the molecules are bent. Deglycosylation of IRBP (with peptide:N-glycosidase F) results in a somewhat smaller molecule that retains its rod-like shape, as shown by gel filtration and sedimentation data. The results indicate that IRBP is an elongated molecule and suggest that a structural change may occur upon ligand binding.     

Quaternary structure of the liver microsomal cytochrome P-450

Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies a position at the vertices of a triangular antiprism with a 32 point group symmetry.     

Two-dimensional crystal packing of matrix porin. A channel forming protein in Escherichia coli outer membranes

Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981). Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm). The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72. At the higher ratio, large hexagonal lattices predominated. Higher lipid ratios did not reveal other crystal forms. The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983). The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm. The mass distribution between triplets, however, were significantly different. They are likely to correspond primarily to lipids. Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers. The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis.     

Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia

Scanning transmission electron microscopic analysis revealed that the 14S fraction of Tetrahymena dynein was of a mixture of two types of particles in approximately equal proportions. The 14S dynein molecules were roughly ellipsoid in shape with approximate axes of 9.5 and 14.5 nm. About half of the particles had tails 20-24-nm long. By the integration of electron scattering intensities, particles with tails had an average mass of 510 kD with a SD of 90 kD. The globular heads of both types of particles had an average mass of 330 kD with a SD of 60 kD. The mass of the tail structure was about 180 kD. By SDS-PAGE, the 14S dynein consisted of two high molecular mass polypeptides above 300 kD that could be distinguished by immunoblot analysis.     

Emulsifier of Arthrobacter RAG-1: chemical and physical properties.

The extracellular emulsifier of Arthrobacter RAG-1 was deproteinized by hot phenol treatment and purified by fractional precipitation with (NH(4))(2)SO(4). The active fraction, precipitating between 30 and 35% saturation [EF-RAG(UET) WA], appeared to be homogeneous by immunodiffusion and sedimentation analysis. EF-RAG(UET) WA had an intrinsic viscosity of 750 cm(3)/g, a sedimentation constant of 6.06S, a diffusion constant of 5.25 x 10(-8) cm(2) s(-1), and a partial molar volume of 0.712 cm(3) g(-1). From these data a weight average molecular weight of 9.76 x 10(5) and a viscosity average molecular weight of 9.88 x 10(5) were calculated. EF-RAG(UET)WA contained 46.7% C, 7.01% H, and 6.06% N. Titration of the nonreducing polymer gave a single inflection point (pK' = 3.05), corresponding to 1.5 mumol of carboxyl groups per mg. Direct estimation of O-ester and hexose content of the highly acidic polymer yielded 0.65 and 0.29 mumol/mg, respectively. Mild alkaline hydrolysis released fatty acids with an average molecular weight of about 231. Strong acid hydrolysis of EF-RAG(UET)WA yielded d-glucose (minor), d-galactosamine (major), and an unidentified amino uronic acid (major).     

Isolation and physical characterization of bovine lens crystallins.

The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 X 200 cm) in 0.05 M tris-NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HM alpha, alpha, beta H, beta L, gamma) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 x 10(6), 5.83 x 10(5), 1.58 x 10(5) , 4.59 x 10(4), 2.14 x 10(4) as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight alpha (HM alpha) and alpha, three for beta H, two for beta L and one for gamma. The gross CD patterns of crystallins were about the same in the peptide region (200 nm similar to or approximately 250 nm) with a minimum centered at about 217 nm, indicative of a beta-sheet structure in all crystallins. The [theta] values at 217 nm ranged from --1700 to --3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm similar to or approximately 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.     

Caldesmon from rabbit liver: molecular weight and length by analytical ultracentrifugation

Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 +/- 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 +/- 0.02 S. yields an apparent length of 53 +/- 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-6098).     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNA_f · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.     

Structural determination of the photosystem II core complex from spinach

A photosystem II core complex was purified with high yield from spinach by solubilization with beta-dodecylmaltoside. The complex consisted of polypeptides with molecular mass 47, 43, 34, 31, 9 and 4 kDa and some minor components, as detected by silver-staining of polyacrylamide gels. There was no indication for the chlorophyll-a/b-binding, light-harvesting complex polypeptides. The core complex revealed electron-transfer activity (1,5-diphenylcarbazide----2,6-dichloroindophenol) of about 30 mumol reduced 2,6-dichloroindophenol/mg chlorophyll/h. The structural integrity was analyzed by electron microscopy. The detergent-solubilized protein complex has the shape of a triangular disk with a maximum diameter of 13 nm and a maximum height of 6.8 nm. The shape of this core complex differs considerably from that of cyanobacterial photosystem II membrane fragments, which are elongated particles. The structural differences between both the complexes of higher plants and cyanobacteria are discussed with special emphasis on their association with the antenna apparatus in the photosynthetic membranes.     

Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha

Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha). The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha. Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa). Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.     

Mitochondrial localization and oligomeric structure of HClpP, the human homologue of E. coli ClpP.

A bacterially expressed recombinant HClpP protein, the human homologue of Escherichia coli ClpP protease, was used to obtain specific polyclonal antibodies. Those antibodies identify a 26 kDa polypeptide in mitochondrial subcellular fractions of rat and human liver. Immunofluorescence and electron microscopic studies demonstrate that the mammalian homologue of ClpP is located in the mitochondrial matrix with a tendency to be found in association with the inner mitochondrial membrane. An HClpP recombinant protein with a truncated NH2terminus (missing the first 58 amino acid residues) shows a molecular mass of 26 kDa under denaturing conditions. This N-truncated HClpP recombinant protein shows a native molecular mass of 340 kDa that is identical with the native molecular mass of the partially purified protein from rat liver mitochondria. Electron microscopy shows that the N-truncated recombinant HClpP has a ring shape with seven identical morphological units in the periphery, exhibiting a 7-fold symmetry. The native molecular mass and the electron microscopic studies suggest that mitochondrial ClpP is composed of two heptameric rings with 7-fold symmetry, similar to E. coli ClpP.     

The archaebacterial hypusine-containing protein. Structural features suggest common ancestry with eukaryotic translation initiation factor 5A.

The amino acid hypusine is formed by post-translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF-5A has been known to contain this unique component. We isolated and purified a hypusine-containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius. The mainly cytosolic protein comprised about 0.03% of the post-ribosomal supernatant protein. No other hypusine-containing protein could be detected in S. acidocaldarius. The molar ratio of hypusine/hypusine-containing protein was 1:1. SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF. The N-terminus was blocked. Four cyanogen bromide fragments were partially sequenced and used to derive two 17-base oligonucleotide probes. A 3-kb HindIII fragment of genomic DNA hybridizing with both probes was cloned. By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa. It contained all cyanogen bromide sequences analysed. Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine-containing protein were identical to the nonapeptides centered by hypusine in the three eIF-5A proteins sequenced so far. The Edman procedure gave no phenylthiohydantoin derivative for this position. For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted. In addition, there were a number of conservative changes. The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine-containing protein and eIF-5A. Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function.