Trypanosoma vivax: Absence of host protein on the surface coat

The possible presence of host serum proteins on the surface of Trypanosoma vivax stock Zaria Y486 was studied. Intact washed bloodstream forms from mice were not lysed or neutralized by antisera against mouse serum proteins. Serum against T. vivax prepared in rabbits against an antigen which was a water-soluble trypanosome extract, failed to cross-react with mouse serum when tested by immunoelectrophoresis and immunodiffusion. The T. vivax antigen failed to cross-react with three different anti-mouse sera when tested by the same techniques.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of ¹²⁵I-surface-labeled parasites showed the presence of a cluster of proteins ranging in molecular weights between 57,000 and 45,000 daltons. None of these proteins was precipitated by anti-mouse serum protein sera. The serum against T. vivax precipitated a protein of 50,000 daltons molecular weight.     

Signal peptides and signal peptidase in Drosophila melanogaster

Translation of poly(A)-containing RNA from the female fat body of Drosophila melanogaster in a rabbit reticulocyte cell-free system results in the synthesis of previtellogenin polypeptides (PVs) having higher apparent molecular weights (46,000 and 45,000) than the forms seen after an in vivo pulse labeling. However, when this RNA is translated in the presence of EDTA-stripped microsomal membranes from the dog pancreas, vitellogenin precursors are produced that, upon SDS- polyacrylamide gel electrophoresis, comigrate with the in vivo forms (apparent molecular weights, 45,000 and 44,000). These processed forms are sequestered within the microsomal lumen, as evidenced by their insensitivity to trypsin digestion. Neither processing nor sequestration occur posttranslationally. In addition, a microsomal membrane fraction derived from Drosophila embryos is able to cotranslationally process the PVs as well as a murine pre-light chain IgG. These observations support a signal-mediated mode of secretion in Drosophila, and suggest that signal sequence recognition and signal peptidase activities are conserved even between mammalian and insect systems.     

Effect of calcitonin on the phosphorylation of non-histones from cultured bone cells

32P-uptake into non-histones from bone cell cultures was selectively stimulated in the presence of calcitonin. Comparison of the control and experimental radioactivity profiles of non-histones fractionated by SDS gel electrophoresis showed that, in response to calcitonin stimulation, there was a 2- to 3-fold increase in the specific activity associated with non-histone proteins in the molecular weight range of 10,000 to 45,000 daltons while that of bands between 50,000 to 200,000 decreased.     

Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.     

Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation

Z-DNA affinity adsorption of an Escherichia coli lysate in the presence of excess B-DNA results in a 1000-fold enrichment for three proteins with apparent molecular weights, on SDS/polyacrylamide gel electrophoresis, of 50,000, 90,000 and 100,000. When retention of these proteins on resins constructed with Z-DNA (Br-poly(dG-dC).poly(dG-dC)) was compared with retention on resins constructed with B-DNA or Br-B-DNA, it was found that approximately 100-fold more of the 50,000 Mr protein, 1000-fold more of the 90,000 Mr protein, and greater than 1000-fold more of the 100,000 Mr protein was retained on the Z-DNA resin. No difference in retention on the B-DNA versus brominated B-DNA resin was found, indicating that the increased retention on the Z-DNA resin was not due to bromination of the Z-DNA. This demonstration of Z-DNA-specific binding in vitro makes these proteins candidates for binding to Z-DNA in vivo. In an effort to determine the function of these proteins we have prepared monoclonal antibodies against each protein and isolated its respective gene. Western blot analysis of lysogens carrying these genes confirms their identity and shows that the complete coding region and promoter for each gene has been cloned.     

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.     

Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins.

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.     

Protein antigens of Theileria parva macroschizonts and immune precipitation studies

The proteins of purified macroschizonts from Theileria parva, T. lawrencei, and T. taurotragi were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major proteins of all species had molecular weights of 120,000, 70,000, 65,000, 62,000, 55,000, 44,000, and 35,000. All further experiments were carried out with T. parva. Using 125I surface labelling it was established that proteins with molecular weights of 70,000, 50,000, and 44,000 were membrane constituents. Staphylococcus aureus protein A-mediated immune precipitation studies with 125I-labelled lysates of macroschizonts and a rabbit anti-macroschizont serum specifically recognized proteins with molecular weights of 120,000, 91,000, 70,000, 62,000, and 35,000. A small proportion of sera recovered from Theileria immune cattle specifically recognized proteins with molecular weights of 180,000 and 70,000 in macroschizont-lysates.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

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.     

Stopped-Flow Circular Dichroism Study on Conformational Change of Alpha-Chymotrypsin by Sodium Dodecyl Sulfate

Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [³⁵S]methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispersed cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells.     

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.     

Electrophoretic characterization and immunoblot analysis of the proteins from the myelin-like light membrane fraction of shrimp ventral nerve (Penaeus duorarum)

1. The proteins of the light membrane fraction (LMF) from the ventral nerve of the pink shrimp (Penaeus duorarum) were separated by SDS gel electrophoresis and analysed by staining and immunoblotting. 2. Shrimp LMF carried four major proteins with apparent molecular weights of Mr = 21,500, 40,000, 78,000, 85,000 and four minor components (Mr = 36,000, 41,500, 43,000, 50,000). 3. None of these proteins bound Concanavalin A. 4. The four major proteins showed no reaction with antisera against six vertebrate myelin proteins. Only the minor Mr = 50,000 component was weakly recognized by the antibodies against mammalian myelin P0 protein.     

An investigation of the degradation of cytochrome P-450 hemoproteins using SDS gel electrophoresis.

Treatment with fluroxene or allyl-$\text{iso}$-propylacetamide of rats induced for elevated levels of cytochromes P-450 results in markedly decreased levels of hepatic microsomal cytochromes P-450 and heme as determined by spectral assay but in unchanged levels of cytochromes P-450 as determined by SDS gel electrophoresis. Since SDS gel electrophoresis does not detect changes in the heme of cytochromes P-450, it is concluded that fluroxene and AIA do not chemically degrade the apoproteins of cytochromes P-450.     

Interspecific variations in proteins synthesized by mammalian mitochondria.

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10,000 and 50,000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.     

Identification and characterization of a heterogeneous population of growth hormone receptors in mouse hepatic membranes

Covalent cross-linking of radiolabeled mouse growth hormone (125I-mGH) with the homobifunctional cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) to microsomal membranes prepared from late pregnant mouse liver resulted in the labeling of three specific mGH binding proteins (receptors) with apparent Mr = 125,000, 62,000, and 56,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These same three specifically labeled proteins were present, but with slightly lower apparent molecular weights, when samples were electrophoresed in the absence of reductant. Cross-linking of 125I-mGH to plasma membrane-enriched fractions of late pregnant mouse liver resulted only in the specific labeling of the two lower molecular weight receptors. Removal of all N-linked carbohydrate with peptide: N-glycosidase F resulted in decreasing the apparent molecular weights of the three receptor forms to 110,000, 50,000, and 46,000 for the 125,000, 62,000, and 56,000 molecular weight forms of the receptor, respectively. Smaller decreases in the molecular weights of all three receptor forms were also apparent after treatment with neuraminidase. However, the differences seen in the intact forms of the growth hormone receptor were also present in the deglycosylated receptors. The relationship between the three forms of the growth hormone receptor was further investigated by comparing the fragments produced by proteolytic digestion of the cross-linked receptors with Staphylococcus aureus protease and endoproteinase Lys-C. The fragments produced from all three receptor forms had very similar molecular weights, although there were slight molecular weight differences in the fragments produced by endoproteinase Lys-C digestion. The overall similarity of the fragments produced by the proteolytic digestions suggests that the three forms of the receptor are related.     

Use of streptavidin to detect biotin-containing proteins in plants

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.