Branko Kozulic

N-acetylation of amino sugar methyl glycosides for gas-liquid chromatographic analysis.

Abstract A procedure is described for rapid and quantitative N-acetylation of amino sugars, particularly suitable for gas-liquid chromatographic analysis of constituent carbohydrates in glycoproteins.

Study of the carbohydrate part of yeast acid phosphatase

It has been found that the carbohydrate part of acid phosphatase from yeast Saccharomyces cerevisiae consists of 16 N-glycosidically linked carbohydrate chains containing from 14 to about 150 mannose units. The presence of very small amounts of O-glycosidically linked chains was indicated. Acetolysis studies pointed to a high similarity in the structure of acid phosphatase and mannan carbohydrate chains. A new method is described for cross-linking of acid phosphatase specifically via carbohydrate chains. The possibility to cross-link the enzyme subunits intramolecularly is in accordance with the suggestion that carbohydrate chains play a role in subunit associations.

An apparatus for submerged gel electrophoresis

A novel apparatus for submerged gel electrophoresis is described in detail. It includes an upper buffer compartment, a lower buffer compartment, and a horizontal plate between the two compartments. The horizontal plate is a heat exchanger connected to an external heater/cooler. Buffer circulates between the two compartments through openings in the horizontal plate. In the upper compartment two separated openings are positioned on each side of the horizontal plate between the side walls and long vertical barriers. The barriers initially direct the flow of buffer and define the electric field on the sides of the upper compartment. The electric field is confined essentially into a rectangular box, defined on the ends by the end walls, on the sides by the barriers, on the bottom by the cooling plate, and on the top by the air. Since the volume of buffer is smaller in the electrode compartment than in the reservoir under the cooling plate, this design enables formation of a substantially uniform electric field without creating too high a current. To enhance uniformity of the electric field, anode and cathode consist each of two platinum wires positioned one above the other at a distance of 6 mm. The electrodes can be placed parallel to the sides and perpendicular to the buffer flow or parallel to the ends and the flow of buffer. The stream of buffer in the upper compartment is regulated by two dams, perpendicular to the long barriers, on each end of the horizontal plate.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of endo-N-acetyl-β-d-glucosaminidase H by substrate-affinity chromatography

Endo-N-acetyl-beta-D-glucosaminidase H (Endo H) was purified to homogeneity (3000-fold) from a culture filtrate to Streptomyces plicatus. The key step was substrate-affinity chromatography, which afforded a 1000-fold purification and yielded a protease- and exoglycosidase-free preparation of Endo H. Proteins from the crude sample were applied to the substrate-affinity column, consisting of yeast-invertase glycopeptides bound to Sepharose-immobilized concanavalin A. After washing off the unbound proteins, Endo H was quantitatively eluted by methyl alpha-D-mannopyranoside. Various conditions were tested to achieve an optimal binding of Endo H to this substrate-affinity gel. After substrate-affinity chromatography, Endo H was separated from the coeluted gylcopeptide substrate and some protein impurities by gel filtration and hydrophobic chromatography.

Hydrophilic and Amphiphatic Monomers and use of their Gels as Separation Media

Abstract In this contribution we describe some results of the work carried out by Professor Klaus Mosbachs group at the Swiss Federal Institute of Technology and later at Elchrom. Synthesis of new acrylic monomers was required for the protein imprinting project and, after the finding that N -acryloyl-tris(hydroxymethyl)aminomethane gels offer advantages for electrophoresis, several series of monomers were synthesized and used to prepare separation media. Particularly useful were hydrophilic monomers based on amino sugar alcohols and corresponding amphiphatic monomers that enable separation of proteins by hydrophobic interaction electrophoresis.

ACID PHOSPHATASE, A GLYCOPROTEIN OF THE YEAST CELL WALL

ABSTRACT In the yeast cell wall a number of hydrolytic enzymes are located. Some of them were found to be glycoproteins. Our interest was centered on acid phosphatase, an inducible enzyme of the yeast Saccharomyces cerevisiae. The enzyme was purified to a high degree, and it was found that it contained about 50% of covalently bound carbohydrates (95% mannose with traces of glucose and 5% N-ace-tylglucosamine). The enzyme preparation contained a whole range of molecules, having the same protein part and differing in the carbohydrate content. It was shown that the reason of this difference was not the various number of carbohydrate chains per molecule, but rather different lenghts of these chains. The molecular weight of the enzyme was found to be 286 000 and it consisted of two identical subunits. Amino acid analysis showed that the enzyme contained the greatest percentage of aspartic and glutamic acids and a small quantity of basic amino acids. This indicated an acidic character of the molecules, what was further confirmed by isoelectric focusing; the major enzyme fraction was isoelectric at pH 4.25. The composition and structure of the isolated glycopeptides from acid phosphatase were also studied. The obtained results indicated that the structure of sugar chains was closely related to the yeast mannan structure, and that the carbohydrate parts of acid phosphatase and invertase were very similar.