A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.     

Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis

Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315-1320. 1966.-Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form.     

Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis

Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

An investigation by ion-exchange chromatography of a chromium,amino acid and nicotinic acid mixture

The mixture of chromium, nicotinic acid and the amino acids glycine, glutamic acid and cysteine which stimulates the rate of CO₂ production in a yeast bioassay system was subjected to the separation scheme based on ion-exchange chromatography which has been used to separate the chromium- containing fractions in brewer's yeast, [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., 18, 195 (1983)]. Four chromium-containing fractions (C2 to C5) were obtained by salt gradients and two further fractions (G1 and G2) were obtained using a pH gradient. All were amino acid-containing complexes of chromium and all except C5 also contained nicotinic acid. However, none of the isolated chromium fractions showed any activity in a yeast bioassay. On the basis of previous work, the activity of the original mixture was attributed to the presence of an oxygen-coordinated trans chromium(III)-dinicotinate complex. Biologically- inactive chromium complexes such as Cr(glu)₂(H₂O)⁺₂ and Cr(gly)₂(H₂O)⁺₂ after elution by ammonium hydroxide from Dowex 50W-X12 cation- exchange columns, stimulated the rate of CO₂ production in the yeast bioassay. Elution with other bases, such as lithium hydroxide, potassium hydroxide and sodium hydroxide led to inactive fractions in all cases. A warning is therefore given that the use of ammonium hydroxide-elution of ion-exchange columns to isolate glucose tolerance factor fractions from biological samples (such as brewer's yeast) can lead to active fractions which do not relate to the native material.     

Lipid composition of Sporendonema epizoum

Triglycerides, free fatty acids, free and esterified ergosterol, Q₉, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidylethanolamine, and three different acylglycoses were identified in the soluble lipids of Sporendonema epizoum mycelium. The same compounds as well as a sterol glycoside were also found in conidia. The mycelium is richer than the conidia in phospholipids, Q₉ and free and esterified ergosterol but contains less glycolipids. The most abundant fatty acid in all non-polar fractions is C_18:2. The prevalent fatty acid of the phospholipids is C_18:1, except for conidial phosphatidylethanolamine and mycelial lysophosphatidylethanolamine.     

Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases

Endogenous acceptors for N-acetylglucosamine (GlcNAc), galactose (Gal) or sialic acid (NeuAc) transfer were labeled to high activities when purified hepatic Golgi fractions were incubated with the corresponding radiolabeled nucleotide sugar in the absence of detergent. The in vitro conditions which were optimal for the endogenous glycosylation of GlcNAc and Gal acceptors (Mn²⁺, ATP) also promoted fusion within a subset of Golgi membranes. Electron microscope radioautography revealed that the majority of NeuAc acceptors were associated with unfused Golgi membranes, whereas the majority of Gal acceptors were localized to fused membranes. GlcNAc acceptors were approximately equally distributed between fused and unfused membranes. Under conditions in which Golgi membrane fusion was absent (− Mn²⁺), only NeuAc transfer was active. The majority of endogenous NeuAc acceptors were consequently assigned to the more trans regions of the hepatic Golgi apparatus as concluded from a combination of radioautography (NeuAc transfer) and acid NADPase cytochemistry (reactive medial and trans Golgi saccules). The distribution of NeuAc and Gal transferases was assessed after Percoll gradient centrifugation of disrupted Golgi fractions. The median density of NeuAc transferase was lower than that of Gal transferase. The studies are indicative of distinct Golgi components harboring the majority of acceptors and enzymes for terminal glycosylation.     

Protein Content and Amino Acid Composition of Certain Fungi Evaluated for Microbial Protein Production

The protein and total amino acid contents of four mycelial fungal strains and one yeast were approximately the same for cultures harvested in the mid-log and early stationary growth phases. It was found that Fusarium oxysporum and Fusarium moniliforme contained approximately 30% more protein and total amino acids than Aspergillus niger. The amino acid composition of mycelial protein compares favorably with that of British Petroleum yeast protein Toprina produced commercially on hydrocarbon substrates. Fusarium spp. may be suitable for commercial production of microbial protein, especially when low-cost agricultural or industrial waste products are readily available as energy sources. Genetic manipulation of these fungi, such as induction of mutant strains through irradiation, may be desirable to obtain a mycelial product of improved yield and/or quality.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

Hydroxamic Acid from Histoplasma capsulatum That Displays Growth Factor Activity

Growth factor(s) present in a spent liquid medium after culture of the yeast form of Histoplasma capsulatum enhanced both yeast and mycelial growth of nine isolates tested. Hydroxamic acid extracted from the culture fluid displayed growth factor activity.     

Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides

Carbohydrate-protein linkages of three types are found in the connective tissue proteoglycans; these linkages involve the following monosaccharide-amino acid pairs: xylose-serine; N-acetylglucosamine-asparagine; and N-acetylgalactosamine-threonine (or serine). The biosynthesis of carbohydrate groups containing linkages of the latter two types presumably occurs by the same pathways that have been well established for many glycoproteins, but details of these processes as they pertain to proteoglycans are not yet known. Initiation of polysaccharide chains linked by the xylose-serine linkage takes place by direct transfer of xylose from UDP-xylose to the hydroxyl groups of specific serine residues in the core proteins of the respective proteoglycans, and the xylosyltransferase catalyzing these reactions has been detected in the rough endoplasmic reticulum of embryonic chick chondrocytes. Although the completed or nascent core proteins are the natural substrates for xylose transfer in the intracellular assembly of proteoglycans, a survey of potential exogenous substrates has shown that small peptides containing alternating serine and glycine residues may also serve as acceptors in this reaction. Nevertheless, larger substrates are preferred, such as chondroitin sulfate proteoglycan, which has been deglycosylated by Smith degradation or HF treatment, or silk fibroin, which contains Ser-Gly pairs. In contrast to the sulfated polysaccharides, which are synthesized by carbohydrate transfer to protein in the endoplasmic reticulum and the Golgi apparatus, hyaluronic acid is formed in the plasma membrane by a different mechanism. The reaction by which chains are initiated is not yet known, but recent work by Prehm suggests that this process occurs either by transfer of the glucuronosyl component of UDP-glucuronic acid to UDP-N-acetylglucosamine or by the converse reaction, i.e., transfer of the N-acetylglucosaminyl unit of UDP-N-acetylglucosamine to UDP-glucuronic acid.     

Host-Pathogen Interactions: X. Fractionation and Biological Activity of an Elicitor Isolated from the Mycelial Walls of Phytophthora megasperma var. sojae

An elicitor of phytoalexin production in soybean (Glycine max L.) tissues was isolated from purified Phytophthora megasperma var. sojae mycelial walls by a heat treatment similar to that used to solubilize the surface antigens from the cell walls of Saccharomyces cerevisiae. The wall-released elicitor is a discrete, minor portion of the P. megasperma var. sojae mycelial walls. The elicitor released from the mycelial walls was divided by diethylaminoethylcellulose and concanavalin A-Sepharose chromatography into four fractions, each having different chemical characteristics. The four fractions were obtained from each of the three races of P. megasperma var. sojae. The corresponding fractions from each of the three races are very similar in composition and elicitor activity. The results suggest that the elicitor activity of each fraction resides in the glucan component of the fraction. Evidence is presented to demonstrate that the elicitors are not race-specific and that the accumulation of glyceollin is not sufficient to account for race-specific resistance.     

Dimorphism-associated variations in the lipid composition of Candida albicans

Yeast and mycelial forms of Candida albicans ATCC 10231, growing together in 12 h and in 96 h cultures, were separated and their lipids were extracted and characterized. The total lipid content of the yeast forms was always lower than that of the mycelial forms. In 12 h cultures the lipids from the two morphological forms consisted mainly of polar compounds, viz, phospholipids and glycolipids. In 96 h cultures both the yeast and mycelial forms accumulated substantial amounts of apolar compounds, mainly steryl esters and triacylglycerols. The mycelial forms were more active than the yeast forms in this respect. Major differences in the lipid composition between the two morphological forms involved the contents of sterols and complex lipids that contain sterols. As a rule, the yeast lipids contained much larger proportions of free sterols than the mycelial lipids. However, the mycelial lipids contained several times more sterols than the yeast forms but bound as steryl glycosides, esterified steryl glycosides and steryl esters. Steryl glycosides and esterified steryl glycosides occurred in yeast lipids only in traces, if at all. The major steryl glycoside in the mycelial forms was unequivocally identified as cholesteryl mannoside. At both phases of growth the apolar and polar lipid fractions from the mycelial forms contained higher levels of polyunsaturated fatty acids (18:2 and 18:3) but lower levels of oleic acid (18:1) than the corresponding fractions from the yeast forms. The lipid content and composition of 12 h and 96 h yeast and mycelial forms of C. albicans KCCC 14172, a clinical isolate, were almost identical with those of C. albicans ATCC 10231.     

Ribosomal RNA from the yeast and mycelial phases ofHistoplasma capsulatum

Purified yeast and mycelial phase rRNA fromHistoplasma capsulatum was heat-formaldehyde deantured and analyzed by polyacrylamide gel electrophoresis. An average difference of 6.9% was seen in electrophoretic migration between the yeast and mycelial phase denatured 17 S rRNAs that was not apparent for the native 17 S rRNAs. This difference was statistically significant and could not be accounted for by random variation among gels electrophoresed in parallel. A significant difference was not found between the 25 S rRNAs of yeast and mycelial phase cells. Molecular weights of 1.28 × 10⁶ for both the yeast and mycelial phase 25 S rRNA, and 0.79 × 10⁶ and 0.73 × 10⁶ for the yeast and mycelial phase 17 S rRNA, were estimated by using denaturing conditions which minimize conformational contributions to electrophoretic migration in polyacrylamide gels. In addition, we find that both the yeast and mycelial phase 17 S and 25 S rRNAs ofH. capsulatum are smaller than their respective counterparts inS. cerevisiae. The results indicate that a structural difference may exist, at least between the 40 S subunits ofH. capsulatum yeast and mycelial phase ribosomes.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

DNA polymerases from a dimorphic fungus,Histoplasma capsulatum

DNA polymerases from the yeast and mycelial phases of a fungus,Histoplasma capsulatum, were partially purified and analyzed. Two classes of enzymes were identified in each morphological form. No significant differences were found among polymerases Ia and Ib from mycelia and I from yeast; similarly, polymerases II from both phases were almost identical. (Numbering refers to the order of elution from phosphocellulose.) However, all of the class I polymerases differed significantly from the class II enzymes. DNA polymerases I (M_r 122,000) were unaffected by 0.2m KCl and resistant to 10 mmN-ethylmaleimide; their levels fluctuated significantly in relation to cell growth, and they showed no nuclease activity. All of the enzymes had similar template dependence and Mg²⁺ requirements. These results are compared to data on other eucaryotic DNA polymerases. Possible roles for class I and class II polymerases are discussed.     

Measurement of uridine diphosphate glucuronic acid concentrations and synthesis in animal tissues

1. A method for the isolation from animal tissues of UDP-glucuronic acid by one-dimensional paper chromatography is described and its concentrations in some tissues of several species of vertebrates are reported; the incorporation of [³²P]-phosphate into UDP-glucuronic acid in vivo was also investigated. 2. The concentration of UDP-glucuronic acid was higher in the liver of rats, rabbits and guinea pigs than in the same tissue of some species of birds, amphibia and fishes; also, the concentration of UDP-glucuronic acid in rat liver, kidney and small intestine was several times lower than that of the same tissues of guinea pigs. 3. The rate of [³²P]-phosphate incorporation into UDP-glucuronic acid was very high in rat liver and kidney and almost reached equilibrium with the radioactivity of UDP-glucose 30min after the administration of the [³²P]phosphate.     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Biosynthesis of polyuronides inMucor rouxii: Partial characterization of fucosyl transferase

Most of the fucosyl transferase activity fromMucor rouxii was detected in a crude membrane fraction. The enzyme transferredl-fucose from GDP-fucose to endogenous and exogenous acceptors. When crude membrane fractions were treated with neutral detergents such as Trition X-100 or Brij 36 T enzyme activity became dependent on exogenous acceptors such as mucoric acid or mucoran. Brij-treated membrane fractions showed maximum fucosyl transferase activity at pH 6.5, and at a temperature between 22 and 28°C. The cations Mn²⁺, Mg²⁺, Co²⁺, Zn²⁺, Fe²⁺, and Ca²⁺ activated the enzyme about twofold. The former was slightly more stimulatory at 4 mM. Km for GDP-fucose was 10 μM. Evidence was obtained that mucoric acid serves as acceptor for fucosyl moieties. Acid hydrolysis of the product synthesized from GDP-fuc by Brij-treated membrane fractions revealed fucose as the major radioactive sugar.