Interaction of concanavalin a with bacterial lipopolysaccharides in agarose gel

Binding of fluorescein isothiocyanate-labeled concanavalin A to a series of molecular species of lipopolysaccharide (LPS), purified from pathogenic bacteria, was studied via agarose gel precipitation experiments and the results were compared with available structural data.The LPS species could be divided into ConA-reactive and non-reactive ones. Reactivity resided in the O-specific chain of LPS, and binding to the lipid A or core moieties of LPS could not be demonstrated by the present methods. The α-D-glucose or α-D-mannose residues of the repeating O-specific oligosaccharide units appeared to be recognized by ConA, except when blocked by steric hindrance. Specificity of the reaction was verified by inhibition with 2% D-glucose. Binding by bacterium-specific sugar-residues could not be demonstrated.For precipitation to occur, polyvalency was required both for LPS and ConA, and the resulting precipitation appeared to be promoted by hydrophobic interactions between the lipid A moieties of LPS molecules. The LPS species were differently retained by the agarose gel, which can be explained by differences in their micellar structure in aqueous solution. E. coli O83 LPS did not readily diffused in 1% agarose gel, but its precipitation with ConA could be demonstrated either at elevated temperature or mixing it previously with molten agarose (Mancini's arrangement).     

黄附子中糖复合物的初步分析

生药中糖复合物研究是２１世纪生物科学热点之一。本文首次报道了附子中糖复合物的研究结果。我们采用饱和硫酸铵沉淀、乙醇分级沉淀及ＤＥＡＢ－Ｃ３２层析方法,从黄附子中分离出三种组分,通过鉴定发现：组分Ｉ主要为糖蛋白,组分Ⅱ主要含酸性多糖,组分Ⅲ可能是淀粉。此研究结果为全面深入地研究和开发附子提供了参考依据。     

Microdetermination of cholesterol in serum lipoproteins.

A two-step procedure for the microdetermination of cholesterol in serum lipoproteins is compared with cholesterol quantitation after density gradient ultracentrifugation. Serum lipoproteins from 10 mul of serum are separated by electrophoresis on agarose and visualized by precipitation with dextran sulfate--CaCl2. The lipoprotein bands are cut off from the plates, the agarose slices are hydrolyzed by gas-liquid chromatography. The comparison between the two procedures reveals satisfactory correlations for beta-and pre-beta-lipoproteins and total serum. There is excellent recovery of cholesterol in fractionated lipoproteins.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.     

Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence

DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.     

Cell-mediated immunity to viruses measured by the indirect agarose technique of leukocyte migration inhibition

The indirect agarose technique of leukocyte migration inhibition has been used to measure the response of human peripheral blood lymphocytes to several viruses. Using commercially available viral antigens, the indirect assay was found to be more sensitive than the direct agarose technique. Supernatants from cultures of sensitive lymphocytes with virus contained a nondialysable factor which inhibited the migration of polymorphonuclear leukocytes (PMN). Under strict conditions of assay, whereby all culture supernatants were tested together on the same PMN preparation, the degree of migration inhibition obtained in response to mumps virus correlated well with the size of the skin test reaction to mumps. A similar relationship was shown for PPD. A good correlation existed also between the degree of migration inhibition and the lymphocyte transformation response for each of these two antigens.     

Preparation and seric form of rabbit C reactive protein]

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP). The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HLD) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.     

Préparation et forme sérique de la protéine réactive C de lapin

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP).The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HDL) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.     

Function,Structure, and Stability of Enzymes Confined in Agarose Gels

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.     

Histone H1 interacts specifically with certain regions of the mouse alpha-globin gene.

We used fragments of a cloned mouse alpha-globin gene to determine if histone H1 interacts selectively with defined regions of a eukaryotic gene. The use of intact plasmids instead of isolated fragments permitted study of relevant sequences in their superhelical form. Several independent experimental approaches (filter binding, precipitation, binding to protein immobilized on nitrocellulose membranes, and agarose gel electrophoresis of the protein-DNA complexes) were used and the histone-DNA interaction was investigated under both noncompetitive and competitive conditions. Binding to subclones encompassing the 5' end of the gene and the first half of the coding sequence is preferred over binding to other subclones. The expression of the sequence-specific selectivity depends on the ionic strength of the binding reaction; the selectivity is mainly expressed under conditions of non-cooperative binding of the histone to DNA. No correlation is observed between AT content and relative affinity of binding to H1. Evidently, other features of DNA structure are involved in the specific H1 binding.     

Quantitative isolation of DNA restriction fragments from low-melting agarose by Elutip-d affinity chromatography

The use of a disposable affinity column and low-melting-temperature agarose for the quantitative preparation of DNA restriction fragments is presented. After electrophoretic separation of DNA, the band(s) are excised and the DNA/agarose melted in a low-salt buffer. After cooling, the DNA is bound to an Elutip-d affinity column. Fragments are eluted at high salt and concentrated by ethanol precipitation. Recoveries greater than 80% are achieved with purity suitable for most applications in molecular biology.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

荧光Real-time PCR鉴定鸡胸骨总RNA质量

为了更准确地鉴定提取鸡骨总RNA的质量,试验分别用核酸蛋白检测仪、1%琼脂糖凝胶电泳和荧光Real-time PCR检测评价3种不同的方法提取成年鸡胸骨总RNA的质量。结果显示,荧光Real-time PCR可更好地鉴定提取的骨总RNA的质量。用核酸蛋白检测仪、1%琼脂糖凝胶电泳能粗略检测RNA的纯度和完整性,但不能反映提取过程中是否引起基因不均一的减少,所检测的纯度也不能精确反映RNA的反转录效率。     

Copurification of bovine milk xanthine oxidase and immunoglobulin

Xanthine oxidase, isolated from bovine milk, exhibited an A280:A450 nm ratio of 5.0. This ratio is reported to be indicative of highly purified enzyme preparations. Serum from a rabbit hyperimmunized against this enzyme fraction exhibited two precipitation lines when incubated with the protein in agarose double diffusion plates. Serum albumin, beta-lactoglobulin, alpha-lactalbumin, lactoferrin, casein, chymosin, and immunoglobulin were tested for reactivity. The second antigen was identified as bovine immunoglobulin. Commercial preparations of xanthine oxidase also contained immunoglobulin as a contaminant. IgG and IgA were present in Sigma (Grade III) fractions and IgM was identified in Boehringer Mannheim preparations. Immunofluorescent studies indicated that xanthine oxidase antiserum reacted with the capillary endothelium of bovine heart. Absorption of this antiserum with bovine IgG abrogated this reaction. These findings may explain apparent discrepancies between reported immunohistological association of xanthine oxidase in heart capillary endothelial cells and the absence of detectable enzymatic activity.     

Affinity interactions between agarose and β-1,4-glycans: a model for polysaccharide associations in algal cell walls

This paper reviews the extensive and previously unpublished work on the interactions between agarose and 1,4-linked β-d-glycans carried out at Unilever Research, Colworth Laboratory, UK. The effect of the following variables is discussed: (i) galactose content of galactomannans; (ii) substitution patterns in the agarose molecule; (iii) structural variations in the 1,4-β-d-glycan main chain; and (iv) molecular size of the 1,4-β-d-glycans.Double helices of agarose, a non-substituted regular polysaccharide comprising 1,3-linked β-d-galactose and 1,4-linked 3,6-anhydro-α-l-galactose, bind in an ordered cooperative fashion to an extended ribbon ordered conformation of sequences of 1,4-linked β-d-mannopyranose residues in plant galactomannans to give mixed gelling systems. This interaction survives, in a modified form, substitution along the agarose molecule by O-methyl ether and O-sulphate esters at O6 of the d-galactose and O2 of the 3,6-anhydro-l-galactose, and 4,6-linked pyruvic acid ketal groups on the d-galactose. The higher the level of substitution on the agarose, the weaker the interaction with galactomannan.In general, the higher the level of galactose substitution in the galactomannan the lower the extent of interaction with agarose. Evidence is presented, however, which indicates that the fine structural distribution of galactose along the galactomannan molecule is also an important determinant for the co-gelling interaction. Substituted 1,4-linked β-d-glucomannans, β-d-glucans and β-d-xylans which can form closely similar extended ribbon order conformations to the galactomannans also participate in co-gelling interactions with agarose. These β-d-glycans are similar in structure to important skeletal polysaccharides such as hemicelluloses and cellulose. This suggests that the binding between agars and β-d-glycans might mimic biological cohesion between skeleton and gel phases in natural red seaweed cell walls. The sensitivity of the interactions studied to fine details of agar and β-d-glycan structure is what might be expected on biological grounds, since the wide and subtle variations of natural polysaccharide structure are presumed to represent a mechanism for control of their intermolecular interactions.     

Partial purification and characterization of chick-embryo prolyl 3-hydroxylase.

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.     

Enhanced Detection of Australia Antigen in Serum Hepatitis Patients by Discontinuous Counter-Immunoelectrophoresis

The use of discontinuous counter-immunoelectrophoresis enhanced the reaction between Au/SH antigen and its antibody in agarose. The ionic strength of the Veronal buffer used in the agarose was 0.015 mu, whereas 0.075 mu Veronal (both pH 8.6) was used for anode and cathode buffers. Electroendosmosis is increased under such conditions. Au/SH antigen and antibody reacted to give sharp lines within 30 to 45 min as compared with conventional counter-immunoelectrophoresis which required 1 to 3 hr or longer.     

Purification of proteins by fractional interfacial salting out on unsubstituted agarose gels

Proteins can be precipitated onto the surface of unsubstituted agarose at an ammonium sulfate concentration about 10% lower than needed for precipitation out of solution. The protein is fractionally redissolved by developing agarose columns with a linear, decreasing gradient of ammonium sulfate. The method is characterized by high reproducibility, good purification factors and high recovery of enzymatic activity. As an example the method is applied to the purification of aminoacyl-tRNA synthetases (E.C. 6.1.1.-) specific for phenylalanine, isoleucine and valine.