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G Birkenmeier

Publications and source records attributed to G Birkenmeier.

At least 37 records · Page 2Linked to original sources
PubMed1996

Ligand interaction of human alpha 2-macroglobulin-alpha 2-macroglobulin receptor studied by partitioning in aqueous two-phase systems.

Alpha 2-macroglobulin (alpha 2-M) is a major proteinase inhibitor in human blood and tissue. Besides its antiproteolytic potential, alpha 2-M was found to modulate antigen- and mitogen-driven immune responses and cell growth by binding and transporting distinct cytokines, growth factors and hormones. The inhibitor is cleared from circulation by binding to a multifunctional cellular receptor present on different cell types. Alpha 2-M, as well as its receptor, are capable of binding a variety of ligands. In the present study we have applied aqueous two-phase systems to analyze the interaction of IL-1 beta and alpha 2-M receptor to different forms of alpha 2-M. The partition of IL-1 beta was changed by addition of transformed alpha 2-M to the two-phase systems rather than by the native inhibitor. The interaction between IL-1 beta and alpha 2-M was enhanced by divalent cations. In addition, the complex formation between 125I-labelled receptor and alpha 2-M could clearly be demonstrated by partitioning. In the presence of divalent cations, transformed alpha 2-M, in contrast to the native inhibitor, effectively changed the partition of the receptor. However, the observed alteration of the partition coefficient was found to be less compared with the values obtained by partitioning of the receptor in the presence of whole plasma containing the inhibitor in equivalent concentrations. The results indicate that other components of the plasma exist which competitively bind to the receptor but independent of Ca2+-ions.

Animals↗
PubMed1996

Insulin-like growth factor-I and alpha 2-macroglobulin in seminal plasma correlate with semen quality.

Insulin-like growth factor-I (IGF-I) and alpha 2-macroglobulin (alpha 2-M) are believed to be involved in the development of germ cells. IGF-I is mainly controlled by concentrations of human growth hormone (HGH), influences cell proliferation and differentiation and its action is mediated by insulin-like growth factor-binding proteins (IGFBP), placental protein 14 (PP14) and prostate-specific antigen (PSA). alpha 2-M acts as a broad spectrum proteinase inhibitor and a binding protein for many cytokines and hormones, e.g. inhibin and activin. This study was designed to identify concentrations of these molecules in seminal plasma in normal semen samples of healthy men, correlations with semen quality, the relationship of IGF-I and alpha 2-M with factors affecting male fertility, and whether vasectomy influences the concentrations of these molecules. Concentrations of IGF-I and alpha 2-M in human seminal plasma were related to semen quality, basal concentrations of HGH, testosterone, IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 in seminal plasma and to serum concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Commercially available assays were used to analyse 69 semen samples of various quality and 11 post-vasectomy samples. IGF-I concentrations in seminal plasma were significantly correlated with the percentage of morphologically normal spermatozoa (r = 0.748, P = 0.00001) and sperm concentration (r = 0.301, P = 0.011), but negatively correlated with serum FSH (r = -0.506, P = 0.00006) and PSA in seminal plasma (r = -0.388, P = 0.00009). Total alpha 2-M was significantly correlated with sperm count (r = 0.423, P = 0.0005), percentage of progressively motile spermatozoa (r = 0.444, P = 0.00019), quality of motility (sperm motile efficiency, r = 802, P = 0.00001) and straight line velocity (r = 0.411, P = 0.0013). Correlation between the sperm concentration and HGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomy reduced the concentration of total alpha 2-M (P = 0.00008) and HGH (P = 0.0068) in the seminal plasma; IGF-I was also reduced after vasectomy when the total ejaculate amount was considered. Thus IGF-I and alpha 2-M are significant for the germ cell development: IGF-I in the maturation of spermatozoa and alpha 2-M in progressive motility.

Follicle Stimulating Hormone↗
PubMed1996

Transformed alpha 2-macroglobulin as a low-affinity growth hormone-binding protein.

Human growth hormone (GH) forms complexes with the purified proteinase inhibitor, alpha 2-macroglobulin (alpha 2-M). This inhibitor occurs in two different forms in serum, known as native and transformed alpha 2-M. It has been clearly demonstrated, using chromatography and electrophoresis combined with autoradiography, that human GH binds specifically to the transformed inhibitor and not to the native protein. The binding was characterized as being mainly non-covalent and involved specific binding sites present only in the transformed inhibitor molecule. Binding analysis, using an immunoprecipitation technique, revealed that GH possesses two different types of binding sites, with dissociation constants of 0.49 +/- 0.12 mumol/l and 61 +/- 8 mumol/l for the high- and low-affinity binding site, respectively. Distribution analysis of 125I-labelled GH in whole plasma suggests that the hormone is bound to two different proteins: first, to the high-affinity GH-binding protein (GHBP) and, second, to the low-affinity GHBP, identified as transformed alpha 2-macroglobulin.

Carrier Proteins↗
PubMed1995

Interaction of murine monoclonal subunit-specific antibodies with phosphofructokinase-1 from Saccharomyces cerevisiae.

Murine monoclonal subunit-specific antibodies against heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae exhibiting no cross-reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS-denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteolytic degradation. Strong complex formation was found with one alpha-specific antibody. Western blotting employing different enzyme forms allowed to localize epitope sites of the alpha-polypeptide chain. Monoclonal antibodies protect phosphofructokinase-1 against chymotryptic degradation.

Animals↗
PubMed1995

Identification of transformed alpha 2-macroglobulin as a growth hormone-binding protein in human blood.

We have found that the human GH forms complexes with the purified proteinase inhibitor, alpha 2-macroglobulin. This inhibitor occurs in two different forms in human blood, known as native and transformed alpha 2-macroglobulin. It could be clearly demonstrated by chromatography and electrophoresis combined with autoradiography that the GH binds specifically to the transformed inhibitor, but not to the native protein. The binding was characterized as being mainly noncovalent to specific binding sites present only in the transformed inhibitor molecule. Binding analysis using antibody precipitation technique revealed two different classes of binding sites with dissociation constants of 0.49 +/- 0.12 and 61 +/- 8 mumol/L for high and low affinity binding sites, respectively. Distribution analysis of 125I-labeled GH in whole plasma suggested that the hormone is bound to different proteins: 1) to the high affinity GH-binding protein, and 2) to the low affinity GH-binding protein identified as transformed alpha 2-macroglobulin.

Chromatography, Gel↗
PubMed1993

Recognition and separation of isoenzymes by metal chelates. Immobilized metal ion affinity partitioning of lactate dehydrogenase isoenzymes.

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated.

Animals↗
PubMed1993

Production of conformation-specific monoclonal antibodies against alpha 2 macroglobulin and their use for quantitation of total and transformed alpha 2 macroglobulin in human blood.

Monoclonal antibodies against the human proteinase inhibitor, alpha 2 macroglobulin, have been produced by immunizing BALB/c mice with alpha 2 macroglobulin reacted with methylamine. Two antibodies have been characterized in detail with respect to their binding to native alpha 2 macroglobulin and to different derivatives of the inhibitor. The antibody alpha-1 was found to recognize only those forms of the inhibitor which were transformed by reaction with different proteinases or with methylamine. Binding of alpha-1 was mapped to a specific epitope localized within a distance of 138 amino acid residues from the C terminal end of alpha 2 macroglobulin. The C terminal end is assumed to be exposed during the transformation of the inhibitor and harbours the receptor recognition site. The monoclonal antibody alpha-11 was found to bind to all forms of the inhibitor indicating that its epitope is located in a region not involved in major conformational changes of the inhibitor. On the basis of the different reactivity patterns of alpha-1 and alpha-11 two enzyme-linked immunosorption assays were established for quantitation of total and transformed alpha 2 macroglobulin in human blood. The concentration of the two forms have been determined in a population of 114 healthy individuals giving values of 254 +/- 6.6 mg/dl (mean +/- SEM) of total alpha 2 macroglobulin and 1.07 +/- 0.05 mg/dl (mean +/- SEM) of the transformed inhibitor.

Animals↗
PubMed1992

Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid.

A two-step method for the isolation of an IgG1 monoclonal antibody against horseradish peroxidase from hybridoma cell culture supernatant is described. Purification was achieved using an aqueous two-phase extraction procedure in conjunction with thiophilic adsorption chromatography. In an aqueous two-phase system composed of 5% PEG 1540 and 22% phosphate the monoclonal antibody preferentially associates with the PEG-rich top phase whereas proteins such as albumin and transferrin partition into the salt-rich bottom phase. Final purification of the monoclonal antibody was achieved by subjecting the PEG-rich top phase to thiophilic adsorption chromatography. The monoclonal antibody purified to homogeneity retained its specificity for horseradish peroxidase as revealed by polyacrylamide gel electrophoresis and an enzyme-linked immunosorbent assay. The potential of this purification protocol for large scale applications is discussed.

Animals↗
PubMed1991

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model.

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.

Animals↗
PubMed1991

Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.

Antibodies, Monoclonal↗
PubMed1991

Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.

Blood Proteins↗
PubMed1990

Affinity partitioning and extraction of proteins.

Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed. Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported. The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples. The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin. The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.

Blood Proteins↗