Polysaccharides of Crithidia fasciculata: Identification and partial characterization of a cell surface constituent

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of d-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight ≥ 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.     

Isolation of multiple forms of rabbit-adrenal norepinephrine N-methyltransferase

Two molecular forms of adrenal norepinephrine N-methyltransferase have been isolated from the nonparticulate fraction of rabbit adrenal glands by use of hydroxylapatite chromatography. The two forms remain distinct on rechromatography. The results obtained by disc gel electrophoresis suggest that the two forms are charge isozymes.The molecular weight of the isozymes was estimated to be 37,000 on the basis of chromatography of Sephadex G-200. The two isozymes are distinguishable on the basis of their steady-state kinetic parameters, particularly on the basis of the substrate inhibition constants for l-norepinephrine and S-adenosylmethionine.     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Subfragments of the papain solubilized TL antigen.

TL antigen was solubilized from the tumor ASLI (TL. 1,2,3) by papain digestion. The subfragments of 125I-labeled TL were examined by two methods. The first involved immune precipitation followed by electrophoresis on SDS-acrylamide gels. This treatment yielded three bands of molecular weight 39,000 and 19,000, as well as material which migrated with the tracking dye. In the second procedure the papain digested material was partially purified on Sephadex G-200. The active fraction from G-200 was labeled with 125Iodine, mixed with alloantiserum and rechromatographed on G-200. The isolated immune complexes were boiled in SDS and 2-mercaptoethanol, then separated on a SDS-Sephadex G-150 column. Two radioactive peaks were eluted indicating an absence of the 19,000 m.w. component following the latter method of purification.     

Isolation of an inhibitor of ß-glucuronidase from porcine sublingual gland

An inhibitor of ß-glucuronidase was isolated from porcine sublingual gland by successive fractionation of trypsin extracts of the latter on Sephadex G-100, DEAE-cellulose, Sephadex G-200, and DEAE-cellulose. Its purity and homogeneity were established by DEAE-cellulose column chromatography, ultracentrifugation, and electrophoresis on cellulose-acetate membrane. The sedimentation coefficient of the purified ß-glucuronidase inhibitor was 3.75 S (S₂₀⁰, w), and the molecular weight was determined to be 340 000 from Sephadex G-200 column chromatography. The inhibitor contained 17.5% protein, 20.8% total hexoses, 19.9% hexosamine, 21.8% N-acetylneuraminic acid, and 9.6% fucose. The inhibition was non-competitive, and it was completely suppressed by the addition of NaCl, KCl, Na₂SO₄, or CaCl₂, respectively.     

Polysaccharides of crithidia fasciculata. Identification and partial characterization of a cell surface constituent.

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of D-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight greater than or equal to 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.     

Isolation of a human serum protein that inhibits the growth of Cryptococcus neoformans

Human serum at 5 to 10% (v/v) in tissue culture medium RPMI-1640, inhibits the growth of Cryptococcus neoformans by 80 to 93%. Serum fractionated on molecular sieve columns (Sephadex G-200) yielded an active protein fraction. This fraction at 100 μg protein/ml inhibited the growth of C. neoformans by 54%. When an active G-200 fraction was applied to a dye affinity column (Affi-Gel Blue) the fraction with inhibitory activity was bound by the column and was eluted with 1.4 M NaCl in 0.1 M phosphate buffer (pH 7.4). The bound fraction at 62.5 μg protein/ml inhibited C. neoformans growth by 82%. On native polyacrylamide gel electrophoresis (Nu-PAGE) the bound fraction migrated as a major and a minor band. Under the reducing conditions of sodium dodecyl sulfate (SDS)-PAGE the bound fraction yielded 4 prominent bands with MW ranging from 175 kDa to 45 kDa. Purification of the active Sephadex G-200 peak was achieved using an anion exchange column (DEAE-Sephacel). Protein eluted with 0.1 M NaCl had strong anticryptococcal activity (12.5 μg/ml, 79% inhibition), which in SDS-PAGE migrated as a single band with an approximate MW of 85 kDA. This protein appears important in natural host resistance to C. neoformans and polymorphisms or deficiencies may have epidemiologic and diagnostic relevance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protein: Polyanion interaction. The effect of heparin on thetrehalosephosphate synthetase of Mycobacterium smegmatis.

The effect of the polyanion heparin on the trehalose phosphate synthetase of Mycobacterium smegmatis had been studied. In the presence of heparin (0.5 mg/ml), the synthetase shows greatly increased stability when heated at 50 °C for various periods of time as compared to the enzyme in the absence of heparin. Heparin also prevents digestion of the enzyme by trypsin. In the absence of heparin, the synthetase is retained on a Sephadex G-200 column and elutes in an area suggesting a molecular weight of about 40,000–50,000. However, when heparin (0.5 mg/ml) is mixed with the enzyme, the synthetase is excluded from the Sephadex G-200 column and elutes in an area suggesting a molecular weight of greater than 450,000. The trehalose phosphate synthetase was purified by binding it to a column of heparin covalently attached to Sepharose 4B. The synthetase was eluted from this column with a linear gradient of heparin. This enzyme fraction which contained bound heparin showed greatly increased stability at 50 °C, and eluted from the Sephadex G-200 column in an area suggesting a molecular weight of greater than 450,000. These results indicate that heparin, and presumably other polyanions, stabilizes the synthetase to adverse conditions and also causes an association of the enzyme to high molecular weight forms.The synthetase, when bound to the heparin-Sepharose gel, still retained good enzymatic activity. This immobilized enzyme was active with various glucose sugar nucleotides (ADP-glucose, GDP-glucose, UDP-glucose, TDP-glucose) and did not require additional polyanion. The product formed from each of these sugar nucleotides was shown to be trehalose phosphate by a variety of chemical and enzymatic procedures.     

Amylases from aleurone layers and starchy endosperm of barley seeds

Amylases from incubated aleurone layers or from starchy endosperm of barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated using acrylamide gel electrophoresis and analytical gel filtration with Sephadex G-200. Electrophoresis of amylase from aleurone layers yields seven visually distinct isozymes with an estimated molecular weight of 43,000. Because each isozyme hydrolyzes β-limit dextrin azure and incorporates calcium-45, they are α-amylases. On Sephadex G-200, amylase from the aleurone layers is separated into seven fractions ranging in estimated molecular weights from 45,000 to 3,000. Little or no activity is observed when six fractions are subjected to electrophoresis. Electrophoresis of only the fraction with the estimated molecular weight of 45,000 gave the seven isozymes. The amylases are heat labile and cannot be stabilized by the presence of substrate or by the protease inhibitor, phenylmethylsulfonylfluoride. Electrophoresis of amylase from the starchy endosperm yields nine β-amylases. Four of these β-amylases are isozymes with an estimated molecular weight of 43,000. The other five forms of β-amylase represent molecular aggregates of the four basic β-amylase monomers. A dimer, a tetramer, and an octamer of β-amylase can be identified with estimated molecular weights of about 86,000, 180,000 and 400,000, respectively. These estimated molecular weights were confirmed on Sephadex G-200. There are five additional fractions of β-amylase with estimated molecular weights ranging from 30,000 to 4,000. These fractions are not observed electrophoretically.     

Enzymatic Decolorization of Melanoidin by Coriolus sp. No. 20

Coriolus sp. No. 20 decolorized a melanoidin solution, a decrease of about 80% in darkness under the optimal conditions. This decolorization occurred with an intracellular enzyme which was prepared from an extract of integrated mycelia, and required aeration and some kinds of sugars, particularly glucose and sorbose. The fraction with melanoidin-decolorizing activity was collected and purified by DEAE-cellulose and Sephadex G-200 column chromatographies. The optimal pH and temperature were pH 4.5 and 35°C, respectively. The molecular weight was found to be about 200,000 by SDS-gel electrophoresis. The purified enzyme was identified as sorbose oxidase; decolorization proceeded in the presence of oxygen and sugars such as maltose, sucrose, lactose, galactose and xylose, besides glucose and sorbose. Glucose in the reaction mixture was converted to gluconic acid. Melanoidin was suggested to be decolorized by the active oxygen formed.     

Isolation of antiserum against rat transcortin and characteristics of specific antibodies

Repeated chromatography of rat plasma protein on DEAE-cellulose, hydroxylapatite and subsequent gel-filtration through Sephadex G-200 were used to obtain a pure rat transcortin homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of transcortin was about 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Immunization of a rabbit with the homogeneous preparation of rat plasma transcortin caused development of antibodies to transcortin. It was shown that the antibodies of rabbit antisera in the experiments made in vitro and in vivo neutralized 60 and 65% of 3H-corticosterone-transcortin complexes, respectively. Specific antibodies to the transcortin were isolated from the homogeneous fraction of IgG by affinity chromatography on transcortin-sepharose 4B. 125J-labelled antibodies were adsorbed by protein A-sepharose; IgG can be eluted by IM acetic acid as a sharp peak. The SDS-polyacrylamide gel electrophoresis demonstrated that affinity-eluted material contains 25 and 50 kDa polypeptides.     

Properties of chicken erythrocyte delta-aminolevulinate synthase

A procedure is described for preparing a fraction highly enriched for chicken blood delta-aminolevulinate synthase (ALA-S) using animals recovering from acetylphenylhydrazine-induced anemia. 1. Blood cells collected from chickens recovering from anemia were disrupted by nitrogen cavitation, and the mitochondrial fraction was prepared from the cell homogenates. ALA-S was released then from mitochondria by sonication and isolated by a procedure involving gel filtration chromatography on Sephadex G-150, fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephacel, and preparative isoelectric focusing. 2. Electrophoretic analyses under denaturing conditions indicated that the final ALA-S preparation was particularly enriched from a 62,200 Da polypeptide. The enzyme eluted from Sephadex G-200 with an equivalent molecular weight of 115,000; this suggested that active ALA-S was a dimer. 3. ALA-S was most active in the pH range of 7.0-8.0, with an apparent KM of 13 microM for succinyl-CoA and of 4.0 mM for glycine. The activity was inhibited 50% by 30 microM hemin.     

A Cytokinin-binding Protein from Wheat Germ: Isolation by Affinity Chromatography and Properties

A cytokinin-binding protein has been isolated from wheat germ via ammonium sulfate precipitation, carboxymethyl Sephadex chromatography, and affinity chromatography on a column substituted with a derivative of kinetin riboside. On Sephadex G-200, the protein migrated with an apparent molecular weight of 122,000 daltons. The dissociation constant for kinetin was determined by equilibrium dialysis to be 1.2 micromolar; N⁶-benzylaminopurine and N⁶-(Δ²-isopentenyl)adenine were also strongly bound. Little affinity was exhibited toward either cis-zeatin or trans-zeatin.     

Isolation and characterization of two antigens of Corynebacterium hofmannii

Banach, T. M. (University of Manitoba, Winnipeg, Canada), and R. Z. Hawirko. Isolation and characterization of two antigens of Corynebacterium hofmannii. J. Bacteriol. 92:1304-1310. 1966.-A serologically active substance, extracted from sonically treated cells of Corynebacterium hofmannii with hot HCl, produced two precipitin lines by immunodiffusion tests with a hyperimmune homologous serum. Extracts of other species failed to precipitate with the hofmannii antiserum. The active fraction was eluted from a diethylaminoethyl cellulose column in the third adsorption peak at a linear concentration of 0.5 m KCl, and produced two precipitin lines which corresponded in identity to those formed by the acid extract. Separation of the antigens was achieved by rechromatography on a Sephadex G-200 column; the major antigen was designated A; the minor, B. The homogeneity and purity of each antigen was established by immunoelectrophoresis and, in addition, that of antigen A by disc electrophoresis. Biochemical analyses showed that both antigens were composed of a major protein component with polysaccharide and nucleic acid present in an approximate ratio of 17:3:1, respectively. Glutamic acid, aspartic acid, alanine, glycine, valine, and leucine were the main amino acids present. Antigen A contained 17% less protein and 3.5% less carbohydrate than antigen B. The principal sugars of antigen A were identified as arabinose and glucose. The molecular weight, estimated by gradient centrifugation, was 16,500 for antigen A and 21,000 for antigen B.     

In Vitro Gibberellin A(1) Binding in Zea mays L

The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]gibberellin A₁ (GA₁) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the ³H-activity bound to this protein was largely [³H]GA₁ and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA₁. Both biologically active and inactive GAs and non-GAs were able to inhibit GA₁ binding. [³H]GA₁ binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

Isolation and characterization of some of the proteins that shuttle between cytoplasm and nucleus in Amoeba proteus

The Rapidly Migrating Proteins (RMP) which shuttle nonrandomly between nucleus and cytoplasm and equilibrate in approximately equal amounts in each compartment, were isolated from Amoeba proteus by implanting ³H-protein containing nuclei into unlabeled cells and some time later extracting the labeled material from the cytoplasms of such cells. The labeled material was subsequently fractionated by gel filtration in Sephadex G-100 columns. The RMP are soluble in dilute salt solutions and appear as a heterogenous group of molecules, one component of which seems to be a single species of protein accounting for ca. one-third of the RMP fraction. Because of its distinctness this component, called the LR fraction, received the major attention in this study. LR was found to comprise ca. 17% of the aqueous-soluble proteins of the nucleus and ca. 3–4% of the total cell protein. LR has a very low molecular weight as determined, e.g., by its elution from a Sephadex G-100 column. Because of its low molecular weight, LR could be purified by taking advantage of the fact that LR is (1) soluble in a saturated Solution of ammonium sulfate and (2) insoluble in butanol, diethyl ether, and 10% trichloroacetic acid. LR migrates toward the anode as a single band when subjected to electrophoresis on “standard disc” and SDS polyacrylamide gels. It does not enter a gel designed to separate basic proteins (at pH 4.0). When subjected to Sephadex G-25 gel filtration LR migrates through the gel as a single band and elutes from the gel at a position in the middle of the linear separation range that indicates its molecular weight is ca. 2300. The only N-terminal amino acid found in the LR fraction is proline. Evidence is presented to show that LR is not the product of a non-specific breakdown of protein produced during its isolation, but the possibility that it results from the cleavage of a single chemical bond of a larger polypeptide, has not been eliminated. When injected into non-labeled amebae, purified radioactive LR concentrates in the nucleus — just as radioactive RMP concentrates in a recipient cell nucleus when an amino acid-labeled nucleus is implanted into an unlabeled cell.     

Isolation and Some Properties of Glucoamylase from Cephalosporium charticola Lindau

High glucoamylase (α-D-/1 → 4/glucan glucohydrolase, EC 3.2.1.3.) activity was obtained in the cell-free culture fluid of Cephalosporium charticola. Glucoamylase seems to be the only amylolytic enzyme produced by C. charticola. The enzyme, purified on diethylaminoethyl-cellulose, was homogeneous by disc gel electrophoresis. The optimum pH on starch was 5.4, and optimum temperature was 60 C. Starch was degraded more rapidly than several other substrates; maltose was hydrolyzed about one-fifth as rapidly as starch. The molecular weight was 69,000, as determined by Sephadex G-100 filtration. The enzyme is a glycoprotein and contains about 6.6% sugars (mannose and glucosamine).     

Materials with opiate receptor binding activity in bovine testis and ovine pancreas

Two groups of opiate-like materials, one with a molecular weight equal to or greater than 5000 daltons and another with a molecular weight smaller than 5000 daltons as judged by gel filtration on Sephadex G-25, were detected in bovine testes. The existence of opiate-like materials with a molecular weight smaller than 5000 daltons was demonstrated in ovine pancreas. The pancreatic fraction most strongly adsorbed on CM-cellulose possessed the highest opiate receptor binding activity. Bovine testis contained corticotropin-like material(s) which stimulated corticosterone production by isolated rat adrenal cells.     

Isolation and Partial Purification of Cadmium-Binding Protein from Roots of the Grass Agrostis gigantea

A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE-Sephadex A-25. The major cadmium fraction was purified further by gel filtration on Sephadex G-75 in 1 molar KCl buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was 1:2.7. Spectroscopic measurements indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days.