sepharose and 1-6-diaminohexane

Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.

Topics: Animals; Blotting, Western; Carrier Proteins; Chromatography, Affinity; Chromatography, Gel; Diamines; Electrophoresis, Polyacrylamide Gel; Malates; Molecular Weight; Physarum polycephalum; Polymers; Sepharose

Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme synthesizes oligosaccharides containing alpha(1-->4) bonds. We have investigated the covalent immobilization of CGTase onto different activated supports. Silica was aminated and further activated with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6-diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was considered.

Topics: Animals; Catalytic Domain; Cyclodextrins; Diamines; Enzymes; Enzymes, Immobilized; Glucosyltransferases; Humans; Immunoglobulin G; Models, Chemical; Protein Structure, Tertiary; Proteins; Sepharose; Silicon Dioxide; Software; Thermoanaerobacter; Time Factors

The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.

Topics: Animals; Cattle; Chromatography, Affinity; Dermatan Sulfate; Diamines; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Hyaluronoglucosaminidase; Male; Sepharose; Testis