Fractionation of plant chromatin with immobilized RNA-polymerase.

A new heptasaccharide, lacto-N-fucoheptaose has been isolated from human milk. It contains D(+)-galactose, D(+)-glucose, L(?)-fucose and N-acetyl-D-(+)-glucosamine in a 3 : 1 : 1 : 2 ratio. The glucose residue is at the reducing end of the oligosaccharide. Data obtained by partial acid hydrolysis, permethylation and enzymic hydrolysis establish the structure of lacto-N-fucoheptaose as follows:

     

Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.     

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.     

Human placenta beta-galactoside-binding lectin. Purification and some properties

A beta-galactoside-binding lectin was extracted from human placenta homogenate with lactose solution and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. The apparent subunit molecular weight of the lectin was 13,800 and its isoelectric point was about 5. Several saccharides containing D-galactose inhibited the hemagglutinating activity. The lectin resembles other vertebrate beta-galactoside-binding lectins in various biochemical characteristics.     

Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K_m value for hydrolysis of nicotinamide-1,N⁶-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.     

Human testis-specific histone TH2B: Fractionation and peptide mapping

The presence of the nonionic detergent Triton X-100 was shown to improve the resolution of the human TH2B on gel electrophoresis and on gel filtration. Total histones of human testis, including TH2B, were resolved by electrophoresis in 15% polyacrylamide gels containing 0.4% Triton X-100, 1.5 m urea, and 0.9 n acetic acid. Gel filtration on Bio-Gel P-200 in 0.4% Triton X-100, 5.0 m urea, and 0.01 n HCl permitted the purification of human TH2B from human testis and sperm in preparative amounts. The structure of human TH2B so prepared was compared to that of rat TH2B, human H2B, and rat H2B by tryptic peptide mapping. The results showed some similarities between all four proteins, but closer similarity was observed within the germ cell histone (TH2B) group and within the somatic histone (H2B) group than between histones of the same species. In addition, human TH2B and rat TH2B each contained one unique peptide absent from other histones.     

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.     

4-Methylumbelliferyl-glycosides as fluorescence probes of sugar-binding sites on lectin molecules: spectral properties and dependence of fluorescence on polarity and viscosity

The spectral properties of 4-methylumbelliferyl-glycosides (MeUmb-glycosides) were investigated in order to assess their usefulness as probes of the microenvironment of sugar binding sites on lectin molecules. It was shown that the abnormally high values for fluorescence polarization of free MeUmb-glycosides (from 0.07 to 0.251) were due neither to their molecular size nor to the blockade of their movement, but to the short lifetimes (less than 0.55 ns) of the excited state of these compounds. Working essentially with two MeUmb-monosaccharides and one MeUmb-disaccharide (MeUmb-alpha-D-galactopyranoside, MeUmb-beta-D-galactopyranoside, and MeUmb-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D- galactopyranoside) which were solubilized in various solvents, it was demonstrated that solvent polarity and viscosity definitely affected the fluorescence intensity of MeUmb-glycosides. A low-polarity medium reduced this intensity, and high viscosity enhanced it. The implications of these findings are discussed in relation to the variations in the fluorescence intensity of MeUmb-glycosides when these compounds were bound to lectins.     

High-performance liquid chromatography of N,O-acylated sialic acids

A rapid, isocratic high-performance liquid chromatographic method for the analysis of N-acetylneuraminic acid, N-glycolylneuraminic acid, and their O-acetylated derivatives is described. Separation of sialic acids and of other monosaccharides as sugar-borate complexes is achieved on an anion-exchange resin. The sialic acids elute as individual peaks after the other sugars tested. The method allows quantitative determination, for example, of amounts of N-acetylneuraminic acid as small as 10 nmol. On cation-exchange resin sialic acids cannot be differentiated, but can be separated from neutral and amino sugars, allowing the determination of as little as 3 nmol of total sialic acids.     

Stability of immobilized alcohol dehydrogenase prepared using the thermostable enzyme from yeast mitochondria

Alcohol dehydrogenase from yeast was partially purified by heat treatment (70°C, 30 min) and immobilized on porous glass, Enzacryl-TI0 and hornblende. The stabilities of these preparations were studied at 30°C and in the case of Enzacryl-TI0 and hornblende at 50°C also. These stabilities were compared with those of immobilized alcohol dehydrogenase from yeast cytosol. In all cases the mitochondrial enzyme provided the more stable bound enzyme conjugates. However, at 50°C the soluble mitochondrial enzyme was more stable than any of the immobilized derivatives: half-life values were 40, 14 and 8 h for the soluble, Enzacryl-TI0 and hornblende samples, respectively.     

Fractionation and characterization of acidic oligosaccharides and glycopeptides from normal and pathological urines

A general procedure is described for the isolation of urinary acidic oligosaccharides and glycopeptides resulting from catabolism of glycoproteins. This procedure has been applied to normal urine and to urine from patients with diseases of the metabolism, including mucolipidosis and fucosidosis.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenylphosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), γ-glutamyltransferase (EC 2.3.2.2), $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase ($\text{acyl-CoA:1-acylglycero-3-phosphocholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution.These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.     

Reconstitution of the d-glucose transporter of bovine thymocyte plasma membrane: Partial purification of transport activity by chromatography on agarose lentil lectin and agarose ethanethiol

The d-glucose transporter of bovine-thymocyte plasma membrane was partially purified using several procedures in sequence. Dimethylmaleic anhydride extraction removed extrinsic membrane proteins (approximately 50% of the total membrane protein) after which sodium cholate solubilized 40% of the residual protein. Reconstitution of solubilized proteins into phospholipid liposomes indicated a 2.5-fold increase in sugar transport specific activity relative to membrane solubilized without dimethylmaleic anhydride extraction. Detergent removal by gel filtration on G-50 Sephadex resulted in reaggregation of intrinsic membrane proteins. Ultracentrifugation of the reaggregated proteins generated a particulate fraction (pellet 1) which contained about 50% of the total d-glucose transport activity of the preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pellet 1 demonstrated removal of a major band at 68,000 daltons and two minor bands not removed by dimethylmaleic anhydride. The 68,000-dalton protein was not removed by any other method tested. Chromatography of resolubilized pellet 1 on a tandem-bed column of agarose ethanethiol and agarose lentil lectin resulted in a 6-fold increase in transport specific activity of nonabsorbed proteins relative to pellet 1. Approximately 15% of the protein (80–90% of the transport activity) applied to the tandem-bed column was recovered in the nonabsorbed fraction. Sodium dodecyl sulfate-gel electrophoresis of proteins in the nonabsorbed fraction showed apparent enrichment of a diffuse zone at 52,00045,000 daltons. The overall increase in specific activity of the partially purified preparation was about 12-fold relative to unpurified solubilized proteins.     

N.M.R. spectroscopy and calcium binding of sialic acids: N-glycolylneuraminic acid and periodate-oxidized N-acetylneuraminic acid

N.m.r. spectroscopy (¹H- and ¹³C-) of N-glycolylneuraminic acid, and of its interaction product with Ca²⁺ at pH 7, indicated that a 1:1 complex is formed, with a formation constant of 193 M^?1 [compared to 121 M^?1 for N-acetylneuraminic acid (1)]. From analysis of electric-field shifts, an approximate position of the Ca²⁺ ion in the complex is inferred, with the hydroxyl group of the N-glycolyl group providing the additional binding. Compound 1 was oxidized with sodium periodate, and ¹³C-n.m.r. spectroscopy was applied in an attempt to identify the aldehyde formed, and to demonstrate that the loss of the glycerol-1-yl side-chain (carbon atoms 8 and 9) decreases its Ca²⁺ ion-binding capacity.     

A theoretical model for immobilized whole cell enzyme

A method of estimating effectiveness factor for immobilized whole cells is developed by considering microbial cells as microspheres containing enzyme activity dispersed in the gel phase of the support matrix. The proper model equations describing the system are solved and the corresponding effectiveness factors calculated for various bead sizes, and numbers and activities of cells. The cell wall resistance (permeability) is found to be one of most important variables in the system. The model is applied in predicting the experimental data of other investigators.