Characterization and translation of transmissible gastroenteritis virus mRNAs.

Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2.     

Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Proteins and Glycoproteins of Paramyxoviruses: a Comparison of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus

The polypeptides of three paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus) were separated by polyacrylamide gel electrophoresis. Glycoproteins were identified by the use of radioactive glucosamine as a carbohydrate precursor. The protein patterns reveal similarities among the three viruses. Each virus contains at least five or six proteins, two of which are glycoproteins. Four of the proteins found in each virus share common features with corresponding proteins in the other two viruses, including similar molecular weights. These four proteins are the nucleocapsid protein (molecular weight 56,000 to 61,000), a larger glycoprotein (molecular weight 65,000 to 74,000), a smaller glycoprotein (molecular weight 53,000 to 56,000), and a major protein which is the smallest protein in each virion (molecular weight 38,000 to 41,000).     

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus.

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.     

Physical Maps of Autographa californica and Rachiplusia ou Nuclear Polyhedrosis Virus Recombinants

TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.     

Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5'-(gamma-thio) triphosphate.

By SDS-polyacrylamide gel electrophoresis, mitochondrial proteins having covalently-bound flavin were analyzed. Mitochondria were prepared from the liver of rat injected with radioactive riboflavin. Radioactivity was found to be associated with four protein components. Their subunit molecular weights were 91,000, 72,000, 60,000 and 44,000. The first two components exhibited yellowish fluorescence on a gel under ultraviolet illumination. The component of the highest molecular weight seems to be a new protein containing covalently-bound flavin.     

Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation

Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two-dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two-dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [³H]fucose as a precursor or by separating [³⁵S]methionine-labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.     

The purification and molecular characterisation of a putative nicotinic-muscarinic acetylcholine receptor from housefly heads

The purification and characterisation of a (³H)-decamethonium binding component which is a putative acetylcholine receptor with a mixed nicotinic-muscarinic pharmacology is described. The protein fraction obtained after differential centrifugation and gel permeation chromatography bound up to 1.8 μmoles of decamethonium/g protein and was shown to be low in acetylcholine esterase and other contaminating proteins. Preparative isoelectrofocusing of the purified putative receptor fraction produced two protein bands, with pI values of 4.7 and 4.9, which bound 0.65 and 0.1 μmoles of decamethonium/g protein respectively. SDS-polyacrylamide gel elctrophoresis of the purified putative receptor revealed the presence of two glycopeptides with molecular weights of 83,000 and 91,000. Polyacrylamide gel electrophoresis in the absence of SDS also revealed two glycopeptide bands. At least one of the polypeptides was shown to covalently bind the muscarinic antagonist (³H)-propylbenzilylcholine mustard. Cross-linkage of the solubilised protein with various cross-linking reagents produced a protein molecule with a molecular weight between 320,000 and 380,000. The putative acetylcholine receptor was shown to contain 3% by weight of neutral sugars (galactose, mannose and glucose) and between 1.9 and 2.4% by weight of glucosamine. Amino acid analysis of the purified protein indicates that it contains a high proportion of hydrophilic and polar residues and may therefore be a peripheral membrane protein.     

Number and Molecular Weights of Foot-and-Mouth Disease Virus Capsid Proteins and the Effects of Maleylation

Evidence was obtained by gel electrophoresis that foot-and-mouth disease virus (FMDV) type A(12) protein migrates mainly in a zone corresponding to polypeptide(s) approximately 25,000 daltons in molecular weight. Additional minor components were observed, four with molecular weights ranging from 10,000 to 22,500 daltons and one with a molecular weight of 37,500 daltons. The minor components comprised about 10% of the total protein and were present in variable amounts. The 75S empty capsids contained primarily 25,000-, 37,500- and 50,000-dalton zones. These molecular weights were estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate versus proteins of known molecular weight, including poliovirus and vesicular stomatitis virus proteins. Maleylation of the amino residues of FMDV protein solubilized it to about 5 to 10 mg/ml in aqueous, nondenaturing solvents. This permitted molecular weights to be estimated also by gel filtration. Maleylation of 70% of the available amino groups of the FMDV protein produced heat and sodium dodecyl sulfate-stable polymeric aggregates of 10 to 20% of the 25,000-dalton zone. It also resulted in an increase in the molecular weight of this zone by an amount equivalent (ca. 1,000) to that expected from the added maleyl residues.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Analytical characterization of beetroot vacuole membrane

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gurke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.     

Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum

Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

Fish rhabdoviruses: comparative study of protein structure.

Proteins from four fish rhabdoviruses have been studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viruses were: trout viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHN), spring viremia virus of carp (SVC), and the pike fry rhabdovirus (PFR). For the two salmonid viruses (VHS-IHN), gel electrophoresis indicated the proteins, with molecular weights estimated to be 190,000, 80,000, 38,000, 25,000, and 19,000, respectively. The electrophoretic profile of the two other viruses (SVC-PFR) revealed four major proteins with molecular weights of 190,000 80,000 42,000 and 21,000, respectively. In this case a minor component with 50,000 daltons was found. For each virus only one protein was found to be glycosylated, i.e., the one with a molecular weight of 80,000. A major protein (molecular weight between 38,000 and 42,000) was found to be associated with the nucleocapsid. All these results revealed marked similarities in protein structure between the four fish rhabdoviruses and the previously well-characterized members of rhabdovirus group. However, one can distinguish two groups of viruses: the first one is composed of salmonid viruses (VHS and IHN) with a protein structure comparable to that of rabies virus and potato yellow dwarf virus; the second one is composed of carp and pike viruses, having a protein structure very similar to that of vesicular stomatitis virus.     

Stimulation of protein synthesis by a 34 KD DNA-binding protein from human placenta

A 34 KD DNA-binding protein fraction from human placenta stimulated endogenous protein synthesis in rabbit reticulocyte and wheat-germ cell-free systems. Though the synthesis of several proteins were stimulated by the 34 KD protein, a dose-dependent increase of two polypeptides of molecular weights 42,000 and 51,000 were distinctly observed in reticulocyte lysates. The synthesis of the major protein (beta-globin) was not affected by the 34 KD protein. In both hemin supplemented and unsupplemented lysates, the ability of 34 KD protein to stimulate the synthesis of high molecular weight (HMW) proteins was drastically reduced by Mg++ and not by dsRNA.     

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.