Gerhard Kopperschläger

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.

Some kinetic and molecular properties of yeast phosphofructokinase

The effects of pH on substrate affinities are described. In the presence of UTP, acting as non‐inhibiting phosphate donor, the behaviour of the enzyme towards F‐6‐P was co‐operative, with a Hill coefficient of 2.2.

Methods for the separation of lactate dehydrogenases and clinical significance of the enzyme

Lactate dehydrogenase (LDH), an ubiquitous enzyme among vertebrates, invertebrates, plants and microbes was discovered in the early period of enzymology. The enzyme has been dissolved in several distinguishable molecular forms. In mammals, three types of subunits encoded by the genes Ldh-A, Ldh-B and Ldh-C give rise to a selected number of tetrameric isoenzymes. LDH-A4, LDH-B4 and the mixed hybrid forms of the A- and B-subunits are present in many tissues but with certain distribution patterns. LDH-C4 is confined in mammals to testes and sperm. Numerous techniques have been employed to purify, characterize and separate the different forms of the enzyme. This report deals with the main protocols and procedures of purification of LDH and its isoenzymes including chromatographic and electrophoretic methods, partitioning in aqueous two-phase systems and precipitation approaches. In particular, affinity separation techniques based on natural and pseudo-biospecific ligands are described in detail. In addition, basic physico-chemical and kinetic properties of the enzyme from different sources are summarized in a second part, the clinical significance of the determination of LDH in diverse body fluids in respect to the total activity and the isoenzyme distribution in different organs is discussed.

Molekulargewichtsbestimmungen durch polyacrylamidgelelektrophorese unter verwendung eines linearen gelgradienten

A new method for molecular weight determination using polyacrylamidegel electrophoresis in a linear gel‐concentration‐gradient (3–20°) is described. Plotting the log of molecular weights of several standard proteins against distance of migration or against 3 log of rate of n‐dgration reveal linear relationships in ranges of 50.000 – 200.000 or 100.000 – 400.000 Daltons respectively. On this basis, a simple method for molecular weight determination of proteins (accuracy ± 5°) has been devised. The method can also be applied for an individual protein in protein mixtures using specific staining procedures.

Cibacron blue F3G-A and related dyes as ligands in affinity chromatography

Cibacron Blue F3G-A and related dyes are sulfonated polyaromatic compounds which bind with considerable specificity and significant affinity to nucleotide-dependent enzymes and to a series of other proteins. After fixation to appropriate insoluble supports, they find wide application as ligands in affinity chromatography.

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.

Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human α2-macroglobulin

The human proteinase inhibitor, α2‐macroglobulin (α2‐M), inhibits a large number of proteinases. α2‐M‐proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (α2‐M‐R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C‐terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab α‐1) has been described which reacts with the receptor‐recognizable form of the inhibitor, the so called transformed α2‐M (α2‐Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab α‐1. Out of 25 phage clones a heptapeptide sequence (S‐x1‐x2‐D‐x3‐x4‐K) was obtained containing identical amino acids in three positions. A consensus peptide (S‐R‐S‐D‐P‐P‐K) was synthesized and found to displace α2‐Mt from binding to mab α‐1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.

The Crystal Structures of Eukaryotic Phosphofructokinases from Baker's Yeast and Rabbit Skeletal Muscle.

Phosphofructokinase 1 (PFK) is a multisubunit allosteric enzyme that catalyzes the principal regulatory step in glycolysis-the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by ATP. The activity of eukaryotic PFK is modulated by a number of effectors in response to the cells needs for energy and building blocks for biosynthesis. The crystal structures of eukaryotic PFKs-from Saccharomyces cerevisiae and rabbit skeletal muscle-demonstrate how successive gene duplications and fusion are reflected in the protein structure and how they allowed the evolution of new functionalities. The basic framework inherited from prokaryotes is conserved, and additional levels of structural and functional complexity have evolved around it. Analysis of protein-ligand complexes has shown how PFK is activated by fructose 2,6-bisphosphate (a powerful PFK effector found only in eukaryotes) and reveals a novel nucleotide binding site. Crystallographic results have been used as the basis for structure-based effector design.

The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites.

Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.

Affinity chromatography of bovine heart lactate dehydrogenase using dye ligands linked directly or spacer-mediated to bead cellulose

A number of reactive dyes coupled to bead cellulose directly or spacer-mediated has been investigated in respect of their interaction with lactate dehydrogenase (LDH; E.C. 1.1.1.28) from heart muscle. The Procion dyes Red HE-7B and Navy H-ER, as well as the Remazol dyes Brilliant Blue R and Brilliant Red 5-BN directly bound to bead cellulose provide high binding of LDH and the adsorbed enzyme is eluted specifically from these affinity adsorbents in high yield. In contrast, under the same conditions no binding of LDH has been found to the Procion dyes Green H-4G, Yellow HE-3G, Scarlet MX-G and Orange MX-G, although an opposite behaviour was expected from the results of affinity partitioning in aqueous two-phase systems. However, immobilizing these dyes via a spacer generated strong binding of the enzyme to the affinity adsorbent. The influence of the length of the spacer was studied in respect of the binding capacity and the yield of the enzyme specifically eluted. The applicability of Procion Scarlet MX-G-(diaminohexyl)-bead cellulose for the purification of LDH from muscle extract in one chromatographic step was demonstrated.