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Special Issue of Chemico-Biological Interactions, a journal of molecular and biochemical toxicology, Vols. 119–120 (1999) 1–620

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.

The hypothalamus and pituitary are the sites through which androgen and estrogen hormones perform the feedback control of gonadotropic mechanism. The microcalorimetric method was used to study the specific reaction between 5α-dihydrosterone (5α-DHT) and Estradiol (E 2 ) and their receptors in the hypothalamus of male and female rats at the age of 28 d. In order to compare binding affinity, experiments were performed at 4° and 37°C. Molar enthalpy changes for E 2 at 37°C are −43.96 ± 0.453 kcal/mol for female and −25.04 ± 0.222 kcal/mol for male rats, respectively. At 4°C molar enthalpy changes are approximately 50% higher for female and 25% higher for male rats. For 5α-DHT molar enthalpy changes at 37°C are −26.67 ± 1.221 kcal/mol for female and −18.60 ± 0.512 kcal/mol for male rats. At 4°C increase for female rats is over 60% and for male rats 40%. The effect of sex difference is much better expressed for E 2 than for 5α-DHT. Possible binding of E 2 and 5α-DHT to hypothalamus receptors in the female and male rats could be in some connection with the mechanism in regulation of gonadotropic feedback system. Ratios of molar enthalpy changes for E 2 : 5α-DHT are 2:1 in females and 1:1 in males, at 4°C. Difference between these ratios could be an answer for the physiological sex difference.

In order to examine acid phosphatase (EC 3.1.3.2) and ATPase (EC 3.6.1.3) activities of baker's yeast (pH optimum 3.5) a protoplast-secreted enzyme preparation was purified and some physical and chemical properties were studied. Three protein fractions containing ATPase and acid phosphatase activities, in the same ratio as the initial preparation, were separated by ion-exchange chromatography. The first fraction which had the highest protein content yielded a homogeneous preparation after Sepharose 4B chromatogrpahy and was used in further studies. An attempt to estimate molecular weight of this protein was made. Attempts to separate acid phosphatase and ATPase activities by ion-exchange chromatography, gel filtration, isoelectric focusing and sucrose density gradient centrifugation have been unsuccessful. Both activities behaved the same way to heat and urea denaturation. These results suggest that the two activities are associated with the same protein molecule.

The effect of atratone, a selective s-triazine herbicide, on specific reactions in hypothalamus, pituitary and prostate gland was studied. It was found that atratone influenced the biosynthesis of LRF at the hypothalamic level. Increased concentrations of atratone from 2.5 to 8.0 mmol inhibited the synthesis of LRF from 22% to 94%. The presence of atratone (0.4 mmol) in pituitary inhibited the activity of 5α-reductase in experiments conducted in vitro and in vivo (s.c. 0.1 mg of atratone/100 g b.wt.) for approximately the same amount (80%). The presence of atratone inhibited the 5α-dihydrotestosterone (5α-DHT) binding to receptor proteins in rat ventral prostate cytosol. It was found by the microcalorimetric technique that 5α-DHT was bound exothermically to cytosol receptors, while in the presence of 0.4 to 2 nmol/s of atratone the energetic level was changed and the binding was endothermic. In sucrose density gradient separation, the presence of 0.4 or 1 mmol of atratone decreased the binding of 5α-DHT to specific receptors in the 8S fraction, which is further proof of the blocking effect of atratone in the hormone-dependent reactions.

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.

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.

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.

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%.

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-β-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.

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.

The biochemical glucosidation of s-triazines in plant tissue has not been reported so far, although a considerable amount of work has been done to follow the metabolic fate of various biologically active s-triazines. Three main degradative pathways are evident: hydrolysis at C-atom 2, and to a minor extent N-dealkylation at C-atoms 4 and 6, as well as splitting of the triazine ring [l] . At present, only limited results concerning the biochemical hydrolysis of methylthiotriazines within the plant are known. The corresponding hydroxytriazines were detected as the sole metabolites in different plant species. Sulfoxide and/or sulfone derivatives were claimed to be hypothetical intermediates during the removal of the entire methylthio group [2,3], but all our attempts to obtain these compounds by chemical synthesis failed, and we could not even detect their appearance. We found that methylthiotriazines are relatively stable toward hydrolytic attack, but in the presence of an oxidant, hydrolysis proceeds faster with formation of corresponding hydroxytriazine and concomitant formation of dimethyldisulphide, instead of methylmercaptan, which was the main product when hydrolysis occurred in the absence of an oxidant [4]. The same course of the reaction could be followed during the hydrolysis of the di(s-triazinyl) sulphides*. Studying the uptake of 4-chloro-4’, 6-bis(isopropylamino)-6’-ethylamino-di(s-triazinyl) sulphide by cucumber plants (ticumis sativus) we found that this compound was transformed within the tissue into 2-ethylamino-4-S-(/3-D-glucopyranosyl)-6-iso

Durch Umsetzung entsprechender Dichlortriazine (I) und Mercaptotriazine (II) werden die Bis-triazinylsulfide (III) erhalten, die bei der Oxidation mit H2O2, K-permanganat, salpetriger Saure, Monoperphthalsaure, (+)-cis-Monoperoxycamphersaure oder Jodbenzoldichlorid die Dihydroxytriazine (IV) und die Disulfide (V) liefern.

Binding enthalpies of various hormones to bovine serum albumin (BSA) and human serum albumin (HSA) in 50 mM phosphate buffer, pH 7.4, at 37 degrees C have been determined by direct microcalorimetry. The observed enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to BSA were found to be -13.24 plus or minus 0.11 -10.31 plus or minus 0.02, -2.37 plus or minus 0.46, -17.64 plus or minus 0.32 and -17.14 plus or minus 0.36 kcal/mol of hormone, respectively. under the same experimental conditions the enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to HSA were found to be -23.94 plus or minus 0.32, -18.88 plus or minus 0.49, -11.14 plus or minus 0.02, -9.88 plus or minus 0.14 and -20.85 plus or minus 0.39 kcal/mol of hormone, respectively.

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.

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 from yeast Saccharomyces cerevisiae was purified, and its physicochemical and kinetic properties were investigated. The sedimentation coefficient has been determined to be s0(20,w) = 13.6 S. The diffusion constant has been found to be 3.9 X 10(-7) cm2s-1, and the calculated partial specific volume was v = 0.663 cm3/g. From these data, a molecular weight of 252,000 was calculated. Electrophoresis on gel slabs, with a linear concentration gradient of polyacrylamide (4-30%), showed size heterogeneity of the native enzyme preparation and indicated an apparent molecular weight in the range of 170,000 to 360,000. In the presence of sodium dodecyl sulfate, the molecular weight was in the range of 82,000 to 165,000, indicating dimeric structure of the native enzyme, which was confirmed by cross-linking experiments. Isoelectric focusing demonstrated charge heterogeneity of enzyme preparation. From CD spectrum it was calculated that the enzyme contains about 29% of alpha-helical structure. Excitation at 278 nm gave an emission fluorescence spectrum with a maximum at 340 nm. Amino acid analysis revealed a high content of aspartic acid, serine, and threonine. Glycine is found as the NH2-terminal amino acid. Initial velocity dependence on substrate concentration, as well as on pH, and thermostability studies indicated the presence of at least two enzyme forms in the preparation.

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.