scholarly journals Vertebrate lectins, Comparison of properties of beta-galactoside-binding lectins from tissues of calf and chicken.

1979 ◽  
Vol 81 (3) ◽  
pp. 528-537 ◽  
Author(s):  
E B Briles ◽  
W Gregory ◽  
P Fletcher ◽  
S Kornfeld
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thiol Group ◽  
Cross Reactivity ◽  
Cytoplasmic Localization ◽  
Binding Properties ◽  
Fluorescence Studies ◽  
Species Specific

Beta-galactoside-binding lectins were isolated from various calf tissues and from chicken hearts by affinity chromatography on asialofetuin-Sepharose, and were compared with respect to biochemical characteristics, binding properties, antigenic cross-reactivity, and cellular localization. The lectins are all thiol group-requiring, divalent cation-independent dimers, of apparent monomer mol wt 12,000 (calf lectins) or 13,000 (chicken lectin), and acidic pI. The calf lectins appear essentially identical by dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid composition, and radioimmunoassay, while the chicken lectin is distinctly different by these criteria. However, all of the lectins competed for the same binding sites on rabbit erythrocytes, and could be inhibited by the same saccharide haptens (notably lactose and thiodigalactoside). Immuno-fluorescence studies on several cultured cell lines revealed that the bovine and chicken lectins had primarily an intracellular cytoplasmic localization. The beta-galactoside-binding lectins of vertebrates appear to be species-specific rather than tissue-specific.

scholarly journals The effect of salt concentration on the iron-binding properties of human transferrin

1982 ◽  
Vol 201 (3) ◽  
pp. 527-532 ◽  
Author(s):  
J Williams ◽  
N D Chasteen ◽  
K Moreton
Keyword(s):  
Salt Concentration ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Iron Binding ◽  
Iron Release ◽  
Equilibrium Conditions ◽  
Binding Properties ◽  
Kinetics Of ◽  
Terminal Site

The salt dependence of the iron-binding properties of transferrin was studied by urea/polyacrylamide-gel electrophoresis. The distribution of iron between the N-terminal and C-terminal binding sites under equilibrium conditions and the rates of release of iron from the two sites were studied. It was found that salt increases the thermodynamic stability of iron binding in the N-terminal site relative to the C-terminal site. Similar behaviour is observed for the kinetics of iron release, where salt retards the rate of removal of iron from the N-terminal site but facilitates removal from the C-terminal site.

scholarly journals Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues.

1982 ◽  
Vol 92 (1) ◽  
pp. 23-27 ◽  
Author(s):  
E C Beyer ◽  
S H Barondes
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Adult Chicken ◽  
Chicken Tissues ◽  
Adult Tissues ◽  
Embryonic Muscle ◽  
The Many ◽  
Kidney Liver

Two lactose-binding lectins from chicken tissues, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed not significant immunological cross-reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-1 contained only traces of CLL-11 whereas embryonic kidney, a very rich source of CLL-11 contained substantial CLL-1. In both muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-1 in liver and CLL-11 in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-1 and CLL-11 from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found.

EXTRACELLULAR CALCIUM AND PLATELET GLYCOPROTEINS

1987 ◽  
Author(s):  
I Jabbal-Gill ◽  
G I Johnston ◽  
S Heptinstall
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Marked Decrease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Monoclonal Antibody ◽  
Alkaline Ph ◽  
Membrane Glycoproteins ◽  
Crossed Immunoelectrophoresis ◽  
The Stability ◽  
Ca Depletion

Platelet membrane glycoproteins lib and Ilia form Ca++-dependent heterodimer complexes that contain binding sites for fibrinogen and therefore are relevant to the ability of platelets to aggregate together. In this study we investigated the effects of extracellular Ca++ on the stability and expression of IIb-IIIa complexes using a IIb-IIIa complex-specific monoclonal antibody M148. Its specificity was examined using crossed immunoelectrophoresis: the antibody reacted only with intact IIb-IIIa complexes and not with either glycoprotein alone.SDS-polyacrylamide gel electrophoresis of immunoprecipitates of soluble glycoproteins that interacted with Ml48 showed that lib and Ilia were present as complexes in Ca++-depleted media at 25°C, pH7.4. However, Ca++-depletion at 37°C, pH7.4 or 37°C, pH8.7 or 25°C, pH8.7 caused dissociation of the complexThe effect of extracellular Ca++ on the expression of IIb-Illa complexes on the surface of intact platelets was studied by a technique which is based upon indirect binding of M148 using a fluorescent- labelled second antibody (FITC-RAM) and measuring the fluorescence per platelet using the FACS IV cytofluorometer. Intact platelets were incubated in Ca++-depleted media at 25°C, pH7.4 or 37°C, pH7.4 either (i) prior to or (ii) after adding M148. At 25°C increased M148-binding was observed, compared to the value prior to Ca++-depletion. This increased binding could be reversed by adding Ca++ back to the preparation. Under condition (i) at 37°C a marked decrease in M148 binding was observed, which could not be reversed by restoring Ca++, while under condition (ii) at 37°C the results were the same as at 25°C.Our studies demonstrate that (a) Ca++-depletion at 37°C and/or alkaline pH causes dissociation of the Ilb-IIIa complex (b) Ca++ depletion at 25°C possibly alters distribution of the complexes thereby increasing their availability to the antibody and (c) M148 prevents the dissociation of complexes in Ca++-depleted media at 37°C, possibly by holding lib and Ilia together

scholarly journals Serological grouping ofTreponema hyodysenteriae

1990 ◽  
Vol 105 (1) ◽  
pp. 79-85 ◽  
Author(s):  
D. J. Hampson ◽  
J. R. L. Mhoma ◽  
B. G. Combs ◽  
J. I. Lee
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Vaccine Development ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Low Molecular Weight ◽  
Serological Response ◽  
Double Immunodiffusion ◽  
Sds Page ◽  
Treponema Hyodysenteriae

SUMMARYTwo Australian isolates ofTreponema hyodysenteriaewhich did not fit within the current serological grouping system for these bacteria wrere examined by agarose gel double immunodiffusion tests (AGDP). Isolate Vic1 was serologically unique, and we propose that it becomes the type organism for a new sixth serological group ofT. hyodysenteriae(Group F). Isolate Q1 was unusual in that lipopolysaccharide (LPS) extracted from it reacted strongly in AGDP with serum raised against the type organism for serogroup D (A1), and also weakly with serum raised against the type organism for serogroup B (WA1). The nature of this cross-reactivity was examined by using cross-absorbed antisera in AGDP, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.The pattern of serological cross-reactivity between Q1, A1 and WA1 was complex and was not fully defined, but the isolate Q1 apparently shared low molecular weight ‘serogroup’ LPS antigens with A1, and shared higher molecular weight LPS antigens with WA1. On this basis Q1 was designated as belonging to serogroup D, although it was recommended that this be qualified as D (B) to indicate the presence of weak cross-reactivity with serogroup B. Such serological cross-reactivity may have significance in relation to the development of immunity toT. hyodysenteriae. Isolate Q1 may be a potentially useful organism for vaccine development because of its ability to induce a good serological response to LPS of treponemes from both serogroups D and B.

scholarly journals Binding properties of detergent-solubilized NCAM.

1990 ◽  
Vol 110 (3) ◽  
pp. 817-824 ◽  
Author(s):  
A K Hall ◽  
R Nelson ◽  
U Rutishauser
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polysialic Acid ◽  
Binding Properties ◽  
Two Dimensional Gel Electrophoresis ◽  
Homophilic Binding ◽  
Brain Membrane ◽  
Specific Antibodies ◽  
Frog Brain ◽  
Species Specific

An assay has been designed for the identification of NCAM-binding proteins present in an NP-40 detergent extract of brain membranes. This method, which is capable of analyzing both heterophilic and homophilic interactions, uses species-specific antibodies against NCAM in combination with radioiodination, so that after unlabeled chicken and iodinated frog brain membrane proteins were allowed to interact, the chicken NCAM could be specifically isolated by immunoaffinity adsorption. The radiolabeled frog proteins coisolated with chicken NCAM were then characterized by one- and two-dimensional gel electrophoresis in combination with immunoblotting. The only detectable NCAM-binding proteins were identified as the 140- and 180-kD forms of NCAM. The presence and absence of polysialic acid on NCAM did not change the amount or nature of the frog proteins immunopurified under these conditions. As an alternative for detecting heterophilic ligands, a simplified immunoprecipitation method was employed using either iodine or sulfate radiolabels. Again under these conditions only NCAM was detected. These results are consistent with the hypothesis that the major binding protein for NCAM is NCAM itself, and suggest that differences in polysialic acid content do not directly alter the properties of NCAM's homophilic binding site.

scholarly journals The distribution of iron between the metal-binding sites of transferrin human serum

1980 ◽  
Vol 185 (2) ◽  
pp. 483-488 ◽  
Author(s):  
J Williams ◽  
K Moreton
Keyword(s):  
Human Serum ◽  
Metal Binding ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Iron Binding ◽  
Metal Binding Sites ◽  
Fresh Serum ◽  
Marked Preference ◽  
Terminal Site

The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250-256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at −15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.

scholarly journals Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement

1986 ◽  
Vol 233 (3) ◽  
pp. 819-825 ◽  
Author(s):  
J Janatova
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gamma Chain ◽  
Complement Components ◽  
The Third ◽  
Disulphide Bonds ◽  
Reduced State

Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.

Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharose and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding ot Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

scholarly journals Conversion of p-coumarate into caffeate by Streptomyces nigrifaciens. Purification and properties of the hydroxylating enzyme

1972 ◽  
Vol 130 (2) ◽  
pp. 425-433 ◽  
Author(s):  
A. M. D. Nambudiri ◽  
J. V. Bhat ◽  
P. V. Subba Rao
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Thiol Group ◽  
Electron Donors ◽  
Copper Protein ◽  
Purification And Properties ◽  
Relationship Of ◽  
The Relationship ◽  
Mediated Catalysis

1. An enzyme responsible for the conversion of p-coumarate into caffeate was purified 97-fold from Streptomyces nigrifaciens. The enzyme had a molecular weight of 18000 as determined by Sephadex G-100 gel filtration and was homogeneous on polyacrylamide-gel electrophoresis. 2. The preparation exhibited both p-coumarate hydroxylase and caffeate oxidase activities. 3. Stoicheiometry of the reaction indicated a mono-oxygenase-mediated catalysis consuming 1mol of O2/mol of substrate hydroxylated. 4. NADH, NADPH, tetrahydropteroylglutamate or ascorbate act as electron donors for the reaction, ascorbate being inhibitory at higher concentrations. 5. The optimum enzyme activity was at about pH7.7 and 40°C, with an activation energy of 39kJ/mol. 6. Monophenols such as p-hydroxyphenylpropionate, p-hydroxyphenylacetate, l-tyrosine and dl-p-hydroxyphenyl-lactate were also hydroxylated by the preparation, in addition to p-coumarate. 7. The enzyme was a copper protein having 0.38% copper in a bound form. 8. Thiol-group inhibitors did not affect the reaction. 9. The relationship of the enzyme to other hydroxylases is discussed.

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