FRACTIONATION OF SOLUBLE BRAIN GLYCOPROTEINS BY AFFINITY AND HYDROXYLAPATITE CHROMATOGRAPHY

—A separation of soluble brain proteins and Con A-binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue-stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A-binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [³H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [³⁶H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane-bound glycoproteins.     

Incorporation of 3H-Gibberellin A20 in roots of G2 peas

Following application of ³H-Gibberellin A₂₀ (GA₂₀) to roots of G2 pea seedlings and homogenization of the roots, about 3% of the radioactivity in the tissue could be precipitated from a 30,000 × g supernatant with trichloroacetic acid (TCA) (soluble fraction) while about 5% of the radioactivity pelleted at 30,000 × g (particulate fraction). The radioactivity in the particulate fraction was soluble in sodium dodecyl sulfate (SDS), but was not dialyzable and was insoluble in ethanol. Electrophoresis of the soluble fraction gave only one band of radioactivity, while that of the particulate fraction gave multiple bands. Acid hydrolysis of the soluble fraction released radioactivity that ran coincident with acid-treated GA₂₀ on silicic-acid column chromatography. The particulate fraction gave numerous radioactive peaks following acid hydrolysis, two of which were coincident with GA₂₀ and GA₂₉ (hydroxylation product of GA₂₀) on silicic acid chromatography. Treatment of the particulate and soluble fractions with RNase, DNase, and proteases showed a significant solubilization of radioactivity only with the proteases, suggesting that the GA is bound to a proteinaceous macromolecule. Complete proteolytic hydrolyis followed by thin layer chromatography showed 65% of the radioactivity from the soluble fraction running separately from free GAs or the individual amino acids; the particulate fraction gave mainly (60%) free GAs on enzymatic hydrolysis and much smaller amounts (17%) in a position separate from that of the GAs or amino acids. Binding of ³H-GA to protease-sensitive material was obtained with biologically active ³H-GA₂₀ and ³H-GA₁.     

An electrophoretic analysis of Schistosoma mansoni soluble egg antigen preparation

Schistosoma mansoni soluble egg antigen (SEA) has been examined electrophoretically. Sodium dodecyl sulfate (SDS) electrophoresis of SEA reveals an extremely heterogeneous protein composition. At least 18-20 distinct bands stain with Coomassie blue and at least 6 bands stain with periodic acid Schiff (PAS). Four of the PAS-positive bands stain only faintly with Coomassie blue. The estimated molecular weight range for these proteins is between 16,000 and 200,000 daltons. An acid soluble fraction was isolated from SEA which contained 5 of the 6 glycoproteins. An immunoelectrophoretic analysis of SEA reveals at least 5 distinct precipitin arcs when developed with serum from mice infected with S. mansoni for 16 weeks.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers

Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)–agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS–agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS–agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS–agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

The acidic polysaccharide from Palmaria decipiens (Palmariales,Rhodophyta)

Palmaria decipiens, one of the most abundant red seaweeds of the chilean Antarctic, was collected in King George Island. The hot water extract (26% yield) showed by acid hydrolysis to contain xylose, galactose and traces of glucose. Fractionation with cetrimide gave a soluble neutral xylan and an insoluble fraction. The insoluble fraction afforded an acidic polysaccharide that contained 4.8% of uronic acids, 2.8% of sulfate and 18.9% of protein. Polyacrylamide gel electrophoresis showed that it was homogeneous. The GLC and HPLC analysis of the total acidic hydrolysis products showed that the acidic polysaccharide was composed of the neutral sugars galactose and xylose in the molar ratio 8.2:1.0 and of galacturonic and glucuronic acid in the ratio 1.5:1.0. The second-derivative FT-IR spectrum showed the characteristic amide I, II and III bands of proteins. Alkaline cleavage with 0.1 M NaOH indicated the presence of a glycoprotein with O-glycosidic linkage.Results found in this work suggest that the acidic polysaccharide extracted from Palmaria decipiens is an acidic xylogalactan-protein complex.     

Metabolism of benzo[a]pyrene in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 38 h with ¹⁴C-labeled benzo[a]pyrene; autoclaved cultures were used as controls. Metabolites were isolated by a sequential extraction procedure and further studied by chromatography or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The soluble metabolites amounted to 1–2.2% in the case of parsley cells, and 19–28% in the case of soybean cells. These metabolites varied in polarity, some being soluble in organic solvent or aqueous buffer while other metabolite fractions were soluble only in hot aqueous sodium dodecylsulphate. In addition, a significant amount of an insoluble metabolite fraction was isolated from the culture fluid as well as the cellular material of soybean suspension cultures.Abbreviations BP benzo[a]pyrene - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

SDS gel electrophoresis of proteins and glycoproteins from peritrophic membranes of some Diptera

Peritrophic membranes (PM) of larval and adult Calliphora erythrocephala and Sarcophaga barbata contain proteins and glycoproteins which were extracted by 2.5% SDS and 8 M urea from the matrix. The acid mucopolysaccharide moiety of PM which was demonstrated by the carbazole method remained in the insoluble resudues. After SDS electrophoresis the gels were counterstained with PAS and Coomassie blue; the carbohydrate and protein content of the bands were recorded by dual scanning. Besides molecular weight (MW) determination from the migration rate, the MW of some glycoprotein bands of PM were evaluated also from their retardation coefficients. The methods revealed different results indicating anomalous SDS binding and mobility of these glycoproteins in SDS electrophoresis.The glycoprotein patterns of larvae and of adults of Calliphora as well as of Sarcophaga differed markedly. PM of adults of both species contained only one carbohydrate fraction which migrated in the gel according to an apparent MW of about 200.000 daltons. PM of the larvae, however, showed a variety of bands in the range between 30.000 and 80.000 daltons which had binding capacities for the protein as well as for the carbohydrate stain. On the other hand, the patterns of pure protein bands were similar in the larval and in the adult stage. Obviously, the glycoprotein pattern of PM is altered during development according to special requirements. Also the similarities between both species in the larval and in the adult stage point to a special physiological function of the glycoprotein moiety.     

Partial purification and characterization of Schistosoma mansoni soluble egg antigen with Con A-Sepharose chromatography

A crude preparation of Schistosoma mansoni soluble egg antigen (SEA) was subjected to affinity chromatography with concanavalin A (Con A) bound to Sepharose 4B. The resulting Con A fractions (bound and unbound) were characterized with sodium dodecyl sulfate (SDS) gel electrophoresis, immunoelectrophoresis, immunodiffusion, and lymphocyte blastogenesis techniques. In the fraction that did not bind to Con A there were at least two distinct antigens, and there were also at least two distinct antigens in the fractions that did bind to Con A. With SDS polyacrylamide gel electrophoresis, at least 20 distinct protein bands (Coomassie blue staining) and three glycoprotein bands (PAS reactive) were present in the unbound fractions from Con A chromatography. The bound fractions separated into at least six distinct glycoproteins with SDS electrophoresis. Although both the bound and unbound fractions contained precipitating antigens, only the bound fractions were capable of eliciting lymphocyte blastogenic responses.     

Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3–4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [³H]-labeled mucin, was precipitated without carrier. [¹⁴C] galactosamine-labeled AB precipitate was β-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P₂ column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse--labeled for 1.0 hr with [³H] leucine or [¹⁴C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.     

PRESENCE OF A SULFATED POLYSACCHARIDE IN THE EXTRACELLULAR MATRIX OF PLATYDORINA CAUDATA (VOLVOCALES,CHLOROPHYTA)1

Evidence for the presence of a sulfated polysaccharide component within the extracellular matrix of Platydorina caudata Kofoid is presented. In situ staining with alcian blue and toluidine blue O indicates accumulation of a sulfated polysaccharide in the matrix. The entire matrix was readily solubilized by a hot aqueous extraction and a sulfated proteoglycan complex was isolated. Thin-layer chromatography of hydrolysates and infrared analysis and chemical desulfation of the intact molecule indicate that the polysaccharide component is principally an arabinogalactan with ester-linked sulfate groups. Protease treatment of the extract revealed two distinct bands separable on cellulose acetate electrophoresis. The slower moving component was a sulfated glycoprotein while the faster moving component was a sulfated mucopolysaccharide essentially free of protein. This is the first report of specific chemical analyses and electrophoretic separation of a sulfated polysaccharide within the matrix of a member of the Volvocales. The cytochemistry and electrophoretic patterns of the P. caudata preparation are compared with the same type of extract made from Chlamydomonas reinhardtii Dang. The possible evolutionary significance of the electrophoretic patterns is presented.     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Structure of Cotylenol,the Aglycone of the Cotylenins Leaf Growth Substances

A potassium chromate-tolerant bacterium was isolated from activated sludge, and the bacterium was identified as Pseudomonas ambigua G–1. The bacterium tolerated up to 2000 ppm of Cr⁶⁺, 1700 ppm of Cu²⁺ and 200 ppm of Cd²⁺, but did not tolerate Hg²⁺. Chemical analysis indicated 86.5% uptake in the soluble fraction and 13.5% uptake in the insoluble fraction of cells. Chromate uptake distribution in the soluble fraction indicated 28.9% in microsomal fraction and 78.1% in supernatant fraction, Chromate distribution in the insoluble fraction showed 61% in lipid-fraction and 21% in polyphosphate-polysaccharidefraction. Chromate inhibited the syntheses of protein, DNA in soluble fraction and RNA in microsomal fraction.     

Isolation and some properties of soluble and membrane-bound cobalamin binding proteins of Euglena mitochondria

Cobalamin binding activity occurred in the soluble fraction (69%) and the membrane fraction (31%) of Euglena mitochondria. The mitochondrial soluble cobalamin binding protein was purified about 580-fold in a yield of 34%; the membrane-bound cobalamin binding protein was solubilized with 2 M urea and partially purified. Both purified mitochondrial cobalamin binding proteins showed low pH dependency for activity. The pH optima of the soluble and membrane-bound cobalamin binding proteins were in the vicinity of 7.0 and 6.0–8.0, respectively. The K _s values of the soluble and membrane-bound cobalamin binding proteins for cyanocobalamin were 0.3 and 0.9 nM, respectively. Neither mitochondrial cobalamin binding proteins required metal ions for activity, but the activity of the soluble and membrane-bound cobalamin binding proteins was inhibited by 1 mM Mn²⁺, 48% and 89%, respectively. Molecular weight of the soluble cobalamin binding protein was calculated to be 93,000. The physiological roles of both mitochondrial cobalamin binding proteins were discussed on the basis of their properties and location in Euglena mitochondria.Abbreviations Cbl cobalamin - Ado-Cbl 5-deoxyadenosylcobalamin - CN-Cbl cyanocobalamin - Me-Cbl methylcobalamin - OH-Cbl hydroxocobalamin - 2-AMP-Cbl 2-amino-2-methylpropanolylcobalamin     

Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan

While it is generally recognized that misfolding of specific proteins can cause late‐onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry‐based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)‐insoluble conformations during aging in Caenorhabditis elegans. SDS‐insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS‐insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS‐insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

INCORPORATION OF [14C]N-ACETYL NEURAMINIC ACID INTO BRAIN GLYCOPROTEINS AND GANGLIOSIDES IN VIVO1

—Intracerebrally administered [¹⁴C]N-acetyl neuraminic acid was incorporated into brain glycoproteins and gangliosides. Incorporation into both classes of compounds was markedly inhibited by acetoxycycloheximide but incorporation into the soluble glycoproteins of the nerve-ending fraction was inhibited least of all. In contrast to glucosamine and fucose, a relatively small proportion of the injected [¹⁴C]NANA was incorporated.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis