Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6³-α-glucosylmaltotriose, 6²-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.     

Purification and mode of action of low molecular weight β-1,4-glucan glucanohydrolase from Trichoderma koningii

Low molecular weight β-1,4-glucan glucanohydrolase (EC 3.2.1.4) has been purified from the culture filtrate of Trichoderma koningii through a three-step procedure including chromatography on Bio-Gel P-150, DEAE-Sephadex A-50 and SP-Sephadex C-50. The molecular weight of the enzyme was estimated to be 22 000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and the isoelectric point was 4.80. The temperature optimum for activity was about 55°C and the pH optimum was 5.5. Thermostability studies showed that the enzyme was almost completely denatured after a 1 h incubation at 60°C. The mode of action of the enzyme was examined by h.p.l.c. using cellooligosaccharides and their mixtures as substrates. It was revealed that the enzyme has transgloycosylation activity. A hypothetical scheme of cellooligosaccharide degradation by the enzyme is proposed.     

Effects of Alloxan Diabetes and Hypophysectomy on Growth,and Plasma and Liver Free Amino Acids of Rats Fed Excess Glycine Diets

Three chitinases (EC 3.2.1.14) were purified from yam, Dioscorea opposita THUMB, by fractionation with ammonium sulfate, chromatographies on DEAE-Cellulose and DEAE-Sephadex A-50, chromatofocusing and gel filtration on Bio-Gel P-60. The purified enzymes (E-l, E-2 and E-3) showed single bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, and the molecular weights were estimated to be 33,500. The pIs were 4.05 (E-l), 4.0 (E-2) and 3.8 (E-3). All enzymes were glycoproteins and the neutral sugar contents were 3.6% (E-l), 3.6 (E-2) and 0.9% (E-3). The N-terminal amino acids of E-l and E-3 were the same and determined to be histidine. All enzymes hydrolyzed glycolchitin, but not p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside or Micrococcus lysodeikticus cell walls. E-l and E-3 were stable in the pH range of 5 ~ 11, and below 60°C. These enzymes showed two optimum pHs around 3.5 and 8.0 or 8.5 with glycolchitin as substrate.     

Pathogenesis-related proteins of tomato : p-69 as an alkaline endoproteinase

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH₄)₂SO₄ fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0.     

Purification of endo-(1→3)-β-D-glucanases lysing yeast cell walls from Rhizoctonia solani

Three forms of $\text{endo}\text{-(1→3)-β-}\text{g}\text{-glucanases}$ lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of $\text{endo}\text{-(1→3)-β-}\text{D}\text{-glucanases}$ lysing yeast cell walls.     

Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-³H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man₉GlcNAc and Man₈GlcNAc and small amounts of Man₇GlcNAc and Man₆GlcNAc. The pulse-chase sample had predominantly Man₈GlcNAc and much less Man₉GlcNAc, indicating that processing of the Man₉GlcNAc to Man₈GlcNAc had occurred during the chase period. Thus, Man₈GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man₉GlcNAc were identical to those of the lipid-linked Glc₃Man₉GlcNAc₂ donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein.     

Isolation and Sugar Composition of Blood Group H-Like Substance from Seeds of Euonymus Sieboldiana

Blood group H-like polysaccharides were isolated from seeds of Euonymus Sieboldiana by a procedure that included fractionation with (NH₄)SO₄, heat treatment, chromatography on DEAE-cellulose and gel filtration on Bio-Gel P-150. One of the highly purified polysaccharide fractions was composed of arabinose, mannose, glucose, rhamnose, galactose and fucose. Arabinose and mannose were the main components, and their molar ratio was calculated as about 3: 1 by gas chromatographic analysis. An analytical ultracentrifugal experiment revealed that the finally purified H-like substance was close to an homogeneous preparation with a small disperse fraction. This heteropolysaccharide inhibited the haemagglutination of human group O red blood cells and eel anti-H serum minimally at 0.004 mg/ml, reacted also with the eel anti-H serum on an immunodiffusion plate to form sharp precipitin line(s).     

Purification and properties of glucoamylase from sugar beet cells in suspension culture

Glucoamylase and α-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with α-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the α-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of α-mannosidase and α-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained α-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M_r (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K_m value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some α-amylases and the debranching enzyme.     

The proteolytic system of Penicillium roqueforti. V. -- Purification and properties of an alkaline aminopeptidase.

An alkaline aminopeptidase was isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by ammonium sulfate precipitation, filtration on Bio-Gel P-100, chromatography on D.E.A.E.-cellulose and hydroxylapatite, filtration on Bio-Gel P-150 and electrofusing. The purified preparation was homogeneous on polyacrylamide gel electrophoresis at pH 8.5. The molecular weight of the enzyme was estimated to be about 35,000 daltons. The isoelectric point is 4.5. The optimum pH for L-leucine-p-nitroanilide hydrolysis is 8.0. At 35 degrees C the enzyme is stable between pH 6.0 and 7.0. Ethylenediamine tetraacetic acid and a sulfhydryl reagent (p-hydroxymercuribenzoate) inhibit the activity, but the enzyme is insensitive to diisopropylfluorophosphate. Hydrolysis of synthetic peptides shows that the enzyme releases apolar amino acids. Dipeptides are poorly hydrolyzed and Gly in penultimate or N-terminal position causes poor activity. The enzyme is able to cleave the N-terminal Arg-Pro bond of bradykinin.     

A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads

An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL₃ by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL₃ protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focusing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

Purification and comparative characterization of an enolase from spinach

An enolase has been purified to apparent homogeneity, as measured by gel electrophoresis, some 400-fold from spinach (Spinacia oleracea). This is the first plant enolase that has been purified to homogeneity. At moderate ionic strengths, the 5,5-dithio-bis-2-(nitrobenzoate) (DTNB)-or parachloromercuribenzoate-reacted enzyme elutes from a Bio-Gel P-200 column with somewhat greater volumes than the yeast enzyme (M_r = 93,000) indicating a greater size. Its elution volume from Ultrogel in 50% ammonium sulfate, however, suggests it exists as an active monomer (M_r = 47,000). Sodium dodecyl sulfate-gel electrophoresis indicates the subunit molecular weight is 50,000 ± 3,000, like that of yeast enolase.

The enzyme contains 23 ± 4 half-cystines per mole of subunit. Titrations with DTNB in guanidine hydrochloride or nondenaturing media indicate that most of these, if not all, are in the reduced state. Reaction of one or more of the sulfhydryls with DTNB or parachloromercuribenzoate stabilizes the enzyme.

The kinetic parameters of the reaction catalyzed by spinach enolase, as well as the inhibitions by transition metal ions and fluoride, are similar to those properties of the yeast and rabbit muscle enzymes.

     

Purification of xylanase from the wood-rotting fungusTrametes hirsuta

Xylanase (EC 3.2.1.8) was purified from a crude extracellular enzyme of the wood-rotting fungusTrametes hirsuta by a four-step procedure involving precipitation with ammonium sulphate, gel filtration on Bio-Gel P-10, column chromatography on DEAE-Sephadex A-50 and preparative electrophoresis on polyacrylamide gel. The isolated enzyme, electrophoretically homogeneous, was separated from β-xylosidase and other hydrolytic enzymes studied. The analysis of the degradation products of water-soluble (4-0-methylglucurono)-D-xylan from willow by the purified xylanase showed it to be endoxylanase (β-1,4-xylan xylanohydrolase). A sequel to Kubačkováet al., Folia Microbiol.20, 29 (1975).     

Comparison of DT-diaphorases purified from the liver cytosol of untreated, and 3,4,5,3',4'-pentachlorobiphenyl- and 3-methylcholanthrene-treated rats

DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from 3-methylcholanthrene (MC)-treated and untreated rats. Treatments with PenCB and MC resulted in about 8- and 7-fold increases of cytosolic DT-diaphorase activity, respectively. Purification of the enzyme preparations from untreated, and PenCB- and MC-treated rats were conducted by using DE-52, DEAE-Sephadex A-50, hydroxylapatite, and Bio-Gel P-150 column chromatographies. Both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all of the final preparations from the three origins were homogeneous and had the same molecular weight of 59,000, consisting of two subunits with molecular weights of 30,000. Further studies on amino acid composition, Km value, optimum pH, and catalytic activities for various substrates also indicated that both PenCB- and MC-inducible DT-diaphorases were identical with that from the untreated rats. All three DT-diaphorases contained about 2 mol of FAD per mol of enzyme. Partial digestion of the enzymes by trypsin and subsequent analysis by HPLC revealed that the three preparations were indistinguishable. The identity among the three purified DT-diaphorases was finally confirmed by Ouchterlony immunodiffusion employing anti-serum raised against each enzyme preparation.     

Structural studies of the acidic oligosaccharide units from bovine glycophorin

The O-glycosidically-linked carbohydrate units of glycophorin from bovine erythrocyte membrane were released by alkaline borohydride treatment. These oligosaccharides were separated into the neutral fractions and the acidic fractions by ion-exchange chromatography followed by gel filtration. The two acidic fractions (fractions 10 and 13) which have the smallest molecular weight in acidic oligosaccharides, were further purified by gel filtration on Bio-Gel P-4 column. Two acidic oligosaccharides (fractions 10-I and 10-II), heptasaccharides, were separated by gel filtration on a Bio-Gel P-4 column from fraction 10. These structures were determined by methylation analyses, nitrous acid deamination after hydrazinolysis and Smith degradation after desialylation. In addition, the structures were also analyzed by direct-probe mass spectrometry of the permethylated derivatives before and after desialylation. These studies indicated that one of them (fraction 10-I) was NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) GalNAcol and another heptasaccharide (fraction 10-II) was Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) [NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→6)]GalNAcol. Athough another acidic fraction (fraction 13) was obtained as a single peak on a Bio-Gel P-4 column, it appeared to be the mixture of a heptasaccharide, NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3 or 6)[Galβ(1→4)GlcNAcβ(1→6 or 3)]Galβ(1→3)GalNAcol and an oligosaccharide similar to fraction 10-II, by analysis of two products obtained by Smith degradation after desialylation.     

Corn Agmatine Iminohydrolase: PURIFICATION AND PROPERTIES

Agmatine iminohydrolase (EC 3.5.3.12) was purified 7,300-fold from extracts of corn shoots by chromatographic separations on diethylaminoethyl-cellulose, Sephadex G-100, and agmatine-affinity column. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weight estimated by Bio-Gel P-200 was 85,000, and the enzyme seems to be a dimer with identical subunits (molecular weight, 43,000). The isoelectric point determined by gel electrofocusing was 4.7. The optimal pH and temperature for activity were 6.5 and 60 C, respectively. The activation energy was 10.9 kilocalories per mole. High specificity exists for agmatine, the K_m value for agmatine was 1.9 × 10⁻⁴ molar, and the enzyme was present in the cytosol. The enzyme was sensitive to Cu²⁺ and Zn²⁺ and also was inhibited by p-hydroxymercuribenzoate and arcain.     

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134?kDa for G6PD, 107?kDa for 6PGD and 121?kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63?kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13?mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.