Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.
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The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits. (+info)
Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes.
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Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes. (+info)
Isolation and characterization of major intrinsic microsomal membrane proteins.
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Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane. (+info)
Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39.
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The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Seventeen proteins (L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39) were isolated from three of the groups (B60, D60, G60) by ion exchange chromatography on carboxymethylcellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.5 to 15 mg. Eight of the proteins (L9, L11, L13, L21, L22, L35', L37 and L39) had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined. (+info)
Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.
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We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes. (+info)
A lipid modified ubiquitin is packaged into particles of several enveloped viruses.
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An anti-ubiquitin cross-reactive protein which migrates more slowly (6.5 kDa) by SDS-PAGE than ubiquitin was identified in African swine fever virus particles. This protein was extracted into the detergent phase in Triton X-114 phase separations, showing that it is hydrophobic, and was radiolabelled with both [3H]palmitic acid and [32P]orthophosphate. This indicates that the protein has a similar structure to the membrane associated phosphatidyl ubiquitin described in baculovirus particles. A similar molecule was found in vaccinia virus and herpes simplex virus particles, suggesting that it may be a component of uninfected cell membranes, which is incorporated into membrane layers in virions during morphogenesis. (+info)
All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.
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All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex. (+info)
Localization and characterization of curved DNA in the human erythropoietin receptor gene by experimental and theoretical approaches.
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We report here the locations of curved DNA in the human erythropoietin receptor gene. A total of 13 DNA bend sites were mapped by circular permutation assays, appearing at an average interval of 651.2+/-214.6 (S.D.) in the 8-kb region. The bend centers in these 13 bend sites were confirmed by oligonucleotide-based assays where most of these centers had bend angles higher than that shown by (AAACCGGGCC) x (A)20 and lower than that shown by (AAACCGGGCC)2 x (A)10. DNA curvature mapping by TRIF software, which is based on the distribution of dinucleotides, primarily AA and TT, provided a highly accurate prediction for the locations of the bend sites. They showed approximately 20 degrees to 40 degrees of bend angles demonstrated by the oligonucleotide assays and by computer analysis. (+info)