Pavao Mildner

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.

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.

Effects of s-triazine herbicides on hormone-receptor complex formation, 5α-reductase and 3α-hydroxysteroid dehydrogenase activity at the anterior pituitary level

Abstract Experiments were performed to study the effects of s-triazine herbicides, atrazine ∗ and prometryne, on 5α-dihydrotestosterone (5α-DHT) receptor complex formation and on the activity of the 5α-reductase and 3α-hydroxysteroid dehydrogenase system in the anterior pituitary of male rats. By sucrose density gradient separation, it was found that in the presence of 0.4 mmol of atrazine or prometryne, 5α-DHT binding to receptor proteins in pituitary tissue was decreased by 27% and 17%, respectively. In in vitro experiments, the addition of atrazine or prometryne decreases the conversion of testosterone to 5α-DHT and the conversion of 5α-DHT to 5α-androstan-3α,17β-diol (3α-diol) in the anterior pituitary. The concentration in the range of 0.6 to 12 mmol of both herbicides, inhibited the 5α-reductase and 3α-hydroxysteroid dehydrogenase activity from 7–92%. in vivo subcutaneous (s.c.) administration of atrazine and prometryne reduced the 5α-reductase activity in the anterior pituitary. A single dose (0.1 mg/100 g b.w.) of atrazine decreases the amount of the 5α-reduced metabolite by 34%, while the same dose injected twice or a double dose (0.2 mg/100 g b.w.) inhibited by 46%. A single dose of prometryne (0.1 mg/100 g b.w.) does not affect the enzymic activity, while two injections of a single dose or a single injection of a double dose (0.2 mg/100 g b.w.) decreased the 5α-reductase activity by 17%.

Preparation of the stabilized glycoenzymes by cross-linking their carbohydrate chains.

Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.e., the oxidation degree and the concentrations of glycoproteins, cross-linker, and hydrogen ions during the cross-linking reaction. It was found that the quantity and size of polymers increased with oxidation degree and protein concentration and by lowering the pH. When the protein concentration was above and pH below certain values, depending on the glycoenzyme, insoluble polymers formed. The soluble cross-linked polymers retained a high level of original activity, and the minor decrease in specific activity noticed was shown to occur during the periodate oxidation step. The cross-linked glycoenzymes are much more resistant to denaturation by high temperature and by changes in pH, demonstrating the usefulness of this method in preparation of the stabilized glycoprotein derivatives.

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.

Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast.

In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.

Purification and evidence for heterogeneity of acid phosphatase from Saccharomycescerevisiae

Abstract The protoplast-secreted acid phosphatase of yeast Saccharomyces cerevisiae was purified about 60 fold by ultrafiltration, gel filtration and chromatography on DEAE-Sephadex A-25. It was established that the enzyme is free of inactive proteins as well as polysaccharides and contains 48% of neutral sugars. The failure to separate the protein from the carbohydrates by several procedures indicates that the carbohydrate part is covalently linked to the protein. A pronounced heterogeneity of the enzyme with respect to charge as well as to molecular weight was found. The data obtained by gel filtration indicated enzyme heterogeneity in respect to carbohydrate content.

Stabilization of Glycoenzymes by Cross‐linking of Their Carbohydrate Chains

The results presented here demonstrate the potential applicability of the described cross-linking method for preparation of soluble glycoenzyme derivatives. The high level of the retained enzyme activity supports the assumption that this approach is superior to the cross-linking through the protein part. In this study, high mannose-type glycoproteins were used. However, the complex-type glycoproteins that are spread among glycoproteins of higher eukaryotes also can be cross-linked by this procedure because, at the least, the terminal monosaccharide will be oxidized by periodate. Moreover, this approach may be applicable when dealing with partially purified glycoenzymes because the protein impurities are not expected to interfere with the cross-linking reaction. Besides cross-linking, other kinds of chemical modifications of glycoenzymes through the carbohydrate part are possible. This, in turn, could lead to changes in the physicochemical and catalytic properties of the enzymes, thereby opening a new field of glycoenzyme engineering.

Role of glycosylation in secretion of yeast acid phosphatase.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.

Influence of glycosylation on the oligomeric structure of yeast acid phosphatase

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units. Dissociation to dimer was in all cases accompanied with a loss of most of the enzyme activity. The underglycosylated acid phosphatase, with less than eight carbohydrate chains per subunit, secreted from cells treated with moderate tunicamycin concentrations, contained besides octamers a high proportion of the dimers. With decreasing levels of enzyme glycosylation, the proportion of dimers increases and the amount of octamers correspondingly decreases. Furthermore, underglycosylated octamers were found to be significantly less stable than the fully glycosylated ones. This showed that carbohydrate chains play a significant role in the octamer formation in vivo, and in stabilization of the enzyme octameric form.