scholarly journals Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods.

1985 ◽  
Vol 100 (1) ◽  
pp. 276-281 ◽  
Author(s):  
H C Schröder ◽  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibodies ◽  
Gradient Centrifugation ◽  
Brain Extract ◽  
Immunological Methods ◽  
Hybridoma Cells ◽  
Alpha Tubulin ◽  
Highly Active ◽  
Reversible Addition ◽  
Tubulin Tyrosine Ligase ◽  
Rapid Purification

Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha-tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.

scholarly journals Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme

1987 ◽  
Vol 104 (4) ◽  
pp. 1059-1067 ◽  
Author(s):  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Gradient Centrifugation ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Alpha Beta ◽  
Tubulin Tyrosine Ligase ◽  
Carboxy Terminal

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.

scholarly journals Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1415-1423 ◽  
Author(s):  
M Goto ◽  
A Murakami ◽  
K Akai ◽  
G Kawanishi ◽  
M Ueda ◽  
...  
Keyword(s):  
Biological Activity ◽  
Monoclonal Antibodies ◽  
Recombinant Human Erythropoietin ◽  
Hybridoma Cells ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Cells ◽  
Human Erythropoietin ◽  
Rapid Purification ◽  
A Site

Abstract We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = approximately 2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.

scholarly journals Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1415-1423 ◽  
Author(s):  
M Goto ◽  
A Murakami ◽  
K Akai ◽  
G Kawanishi ◽  
M Ueda ◽  
...  
Keyword(s):  
Biological Activity ◽  
Monoclonal Antibodies ◽  
Recombinant Human Erythropoietin ◽  
Hybridoma Cells ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Cells ◽  
Human Erythropoietin ◽  
Rapid Purification ◽  
A Site

We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = approximately 2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.

A rapid purification procedure for pyruvate kinase from the hyphal fungus Aspergillus nidulans

1988 ◽  
Vol 34 (10) ◽  
pp. 1154-1158 ◽  
Author(s):  
Harry C. M. Kester ◽  
Jos H. A. A. Uitzetter ◽  
Leo H. de Graaff ◽  
Jaap Visser
Keyword(s):  
Molecular Weight ◽  
Liquid Chromatography ◽  
Aspergillus Nidulans ◽  
Pyruvate Kinase ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Highly Active ◽  
A Subunit ◽  
Rapid Purification ◽  
Dye Affinity Chromatography

Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45–55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65 000 and an isoelectric point of 4.7, was used to determine the amino acid composition.

Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies

1989 ◽  
Vol 92 (3) ◽  
pp. 519-528 ◽  
Author(s):  
P. Draber ◽  
E. Draberova ◽  
I. Linhartova ◽  
V. Viklicky
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Antigenic Determinants ◽  
Alpha Tubulin ◽  
Domain Specific ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
C And N ◽  
Terminal Domains

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6–11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.

scholarly journals Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Ermioni Kalfopoulou ◽  
Diana Laverde ◽  
Karmela Miklic ◽  
Felipe Romero-Saavedra ◽  
Suzana Malic ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Selection Process ◽  
Multidrug Resistant ◽  
Expression Vectors ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Strains ◽  
Variable Regions ◽  
Content Type

ABSTRACTMultidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide ofEnterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein fromEnterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing againstE. faecalistype 2, and SagA.01 showed 40% killing againstE. faecium11231/6. In addition, both MAbs showed cross-reactivity toward otherE. faecalisandE. faeciumstrains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

Thromboplastin: Nomenclature and Preparation of Protein-Free Material Different From Platelet Factor 3 or Lipid Activator

1958 ◽  
Vol 193 (3) ◽  
pp. 584-592 ◽  
Author(s):  
Eugen R. Hecht ◽  
Min H. Cho ◽  
Walter H. Seegers
Keyword(s):  
Lipid Fraction ◽  
Dry Weight ◽  
Brain Extract ◽  
Rabbit Brain ◽  
Platelet Factor ◽  
Highly Active ◽  
Traditional Sense ◽  
Free Material ◽  
The Brain ◽  
Complete Transformation

A method was developed for obtaining a highly active fraction from rabbit brain tissue which meets all of the requirements of a complete thromboplastin in the ‘traditional’ sense. The material we have isolated is called brain thromboplastin. This lipid fraction contains no protein. It is a complex consisting of sterol, glutamic acid, serine, ethanolamine and probably sphingosine. These components are also found in lipid activator and in the lipid portion of purified platelet factor 3, the latter being a lipoprotein. It is lethal upon intravenous injection in suitable quantity, while purified platelet factor 3 and or lipid activator are not. When the brain thromboplastin is combined with Ac-globulin and calcium ions, purified prothrombin changes to biothrombin, but such a change to thrombin does not occur when purified platelet factor 3 or lipid activator replaces the brain thromboplastin. For complete transformation of prothrombin to thrombin, it is sufficient to have the following proportions given on a dry weight basis: prothrombin 500, brain thromboplastin 80, and Ac-globulin 1. Heating brain thromboplastin to 100°C for 30 minutes or refluxing it with ether for 90 minutes impairs only very little of its activity but refluxing with alcohol for 90 minutes destroys its activity. However, then the material can still function as a procoagulant. The word ‘thromboplastin’ apparently now is utilized in all kinds of ways so that the meaning is diluted extensively. We use the term more specifically. The terms lung thromboplastin, beef lung extract thromboplastin, brain extract thromboplastin and lipid activator are used in terms of carefully considered descriptions related to preparations. To fulfill the requirements for generic designations, the word procoagulant can refer to all those substances and conditions that further the coagulation of blood while anticoagulant is the opposite designation.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

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