Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Biological nitrogen and organic matter removal from tannery wastewater in pilot plant operations in Ethiopia

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of d-fructose to d-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce d-mannitol from d-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form d-mannitol from d-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up d-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM d-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD⁺-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y _{D-mannitol/D-fructose}was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.     

Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids

Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Catabolite Repression of induction of aldose reductase activity and utilization of mixed hemicellulosic surgars in Candida guilliermondii

NADPH-dependent aldose reductase activity induced by d-xylose or l-arabinose was detected in cell-free extracts of Candida guilliermondii, but only negligible activities were observed if d-glucose served as carbon source. The induction of aldose reductase activity on mixed sugars was investigated under resting cell conditions. d-Glucose repressed enzyme induction by d-xylose or l-arabinose to varying degrees, and l-arabinose inhibited enzyme induction by d-xylose. During incubation in a mixture of d-xylose-d-glucose, glucose consumption by cells was fast and simultaneous with d-xylose utilization. l-arabinose consumption was poor when it was present as the only sugar and in a mixture with d-glucose; this pentose depletion occurred only when all hexose was consumed. When d-xylose and l-arabinose were present in a mixture, the consumption of both pentoses was reduced by the presence of the second sugar, although both sugars were consumed simultaneously by cells. The results show that induction of aldose reductase activity and d-xylose utilization by cells of Candida guilliermondii are under control of glucose repression.     

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.     

UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.     

Role of sugars in phosphate transport in baker's yeast

Sugar substrates which depress the intracellular level of inorganic phosphate in baker's yeast (d-glucose,d-fructose,d-mannose, sucrose, as well as maltose andd-galactose after appropriate induction) also make transmembrane flux of phosphate anions possible. Acetate and ethanol, although readily oxidized, as well as nonmetabolized sugars, do not produce the effect. Phosphate uptake in whole cells (but not in protoplasts) is accelerated by preincubation with substrate either aerobically or anaerobically but the actual presence of substrate in the incubation medium is required for transport to take place. Starved cells take up phosphate from the medium with aK _m of 3mm, the half-activation concentration by glucose being 18mm, the amount taken up being constant under given conditions (40 μmol/g dry wt. here). Phosphate-rich cells lose phosphate to the medium in the presence of a suitable substrate. The uptake process is characterized by an activation energy of 13400 cal/mol at 10⁻⁶ m phosphate and of 9400 cal/mol at 10⁻³ m phosphate. The process shows two optima at pH 5.0 and 7.0. A short-lived intermediate of fermentative sugar metabolism is postulated as essential for the translocation of phosphate across the yeast membrane.     

Induction of aldose reductase activity inCandida guilliermondii by pentose sugars

Summary Cell extracts ofCandida guilliermondii grown ind-xylose,l-arabinose,d-galactose,d-glucose,d-mannose and glycerol as sole carbon sources possessed NADPH-dependent aldose reductase activity, but no NADH-dependent activity was detected.d-xylose andl-arabinose were the best inducers of aldose reductase activity. The highest enzyme activity ind-xylose orl-arabinose-grown cells was observed first withl-arabinose followed byd-xylose as substrates of the enzymatic reaction. However, only low activity was found ind-glucose,d-mannose andd-galactose-grown cells, indicating that these carbon sources cause catabolite repression. Enzyme activities induced ind-xylose-grown cells were twice as high as those obtained from the cells under resting conditions. Furthermore, the level of induction of aldose reductase activity depended on the initial concentration ofd-xylose. The present study shows that aldose reductase activity may be efficiently induced by pentose sugars of hemicellulosic hydrolysates and weakly by hemicellulosic hexoses.     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

The presence of an endogenous lectin in early embryos ofXenopus laevis

Summary Xenopus laevis embryos were examined for the presence of endogenous carbohydrate binding proteins. Soluble extracts of cleavage, gastrula and neurula embryos are able to agglutinate trypsinized rabbit erythrocytes. Unlike other embryonic lectins this agglutination activity requires the presence of calcium ions but not of sulphydryl reducing agents. It is specifically inhibited by galactose and galactose containing derivatives. Thiodigalactoside is the most potent disaccharide inhibitor followed by lactose and melibiose respectively. Methyl -d-galactopyranoside is a more effective inhibitor than its anomer. N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, methyl -d-mannopyranoside andl-fucose do not inhibit activity at concentrations at or above 25 mM. EDTA and EGTA are also strong inhibitors of this activity. -d-galactoside binding lectins present in the early chick embryo have been implicated in cell to cell and cell to substrate adhesiveness of extraembryonic chick endoderm cells. Since cells of the blastula inXenopus laevis possess surface receptors bearing terminal -d-galactoside groups it is possible that this -d-galactoside binding lectin may play a role in the control of cell surface mediated events during development.     

Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate

Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the d-malate production activity, until the maximal malease activity was reached. Incubation of P. pseudoalcaligenes cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating that cell lysis had occurred. The rate at which the d-malate production activity increased was dependent on the Triton X-100 concentration and on the cell density. Also the rate at which lysis occurred depended on the Triton X-100 concentration.     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Evidence for two β-galactosidase activities inStreptomyces violaceus

The kinetics of β-galactosidase synthesis induced by galactose and lactose inStreptomyces violaceus, as well as the pattern ofo-nitrophenyl-β-d-galactoside-positive bands observed after electrophoresis of both crude extracts, showed the presence of different β-galactosidase activities in the two cellular extracts. It is postulated that the lactase activity induced by lactose is the physiological enzyme responsible for lactose utilization. The possible function of the galactose-induced activity is also discussed.     

Protoplasten der Hefe Rhodotorula gracilis

Zusammenfassung Die Protoplasten der obligat aeroben Hefe Rhodotorula gracilis wurden hinsichtlich ihrer charakteristischen physiologischen und Transporteigenschaften mit intakten Zellen verglichen. Folgende Ergebnisse wurden gewonnen: 1. Endogene und durch d-Glucose stimulierte Atmung entsprach den Werten von intakten Hefezellen. 2. d-Glucose wurde von Protoplasten aus dem Medium aufgenommen und abgebaut. 3. Die Aufnahme von d-Xylose führte zu vielfacher Akkumulation der Pentose im Zellinnern. Nach 50 min wurde ein für den Xyloseabbau induziertes System wirksam. 4. Bei Zugabe im Gemisch wurde die Aufnahme von d-Xylose durch d-Glucose unterbunden. 5. Akkumulierte d-Xylose wurde bei Zugabe von d-Glucose im Austauschtransport durch den mobilen Träger aus der Zelle heraus befördert. 6. Der Zuckertransport, gemessen an der d-Xyloseaufnahme, war streng stoffwechselenergieabhängig und wurde durch Entkoppler vollständig gehemmt.Diese Ergebnisse zeigen, daß die Stoffwechsel- und Transportfunktionen der intakten Hefezellen in ihren Protoplasten vollstädig erhalten bleiben. Die Anwendung von R. gracilis-Protoplasten zur Klärung spezieller Fragestellungen ergab: 1. Der Transport von d-Trehalose erfolgte nach extracellulärer Spaltung des Disaccharides durch Aufnahme der entstandenen Glucose. 2. Densitometrische Messungen an Protoplastensuspensionen zeigten sich geeignet zur kontinuierlichen Aufzeichnung von Zuckeraufnahmevorgängen.

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Protoplasts from the yeast Rhodotorula gracilis II. Physiological and transport properties

The protoplasts of the obligatory aerobic yeast Rhodotorula gracilis (5/Fres/Harrison) were compared with the intact yeast cells with respect to the identity of their physiological and transport properties. It was found: 1. The rates of endogenous and glucose-stimulated respiration of protoplasts were similar to those of the whole cells. 2. d-glucose was taken up from the medium with constant velocity; no free glucose could be detected inside the protoplasts. 3. The uptake of d-xylose led to manifold accumulation of the pentose intracellularly. Within 50 min incubation an enzyme system for the degradation of d-xylose became effective. 4. In a mixture of d-xylose and d-glucose the latter blocked the uptake of the pentose. 5. d-xylose once accumulated was exchanged by the mobile membrane carrier for d-glucose after its addition to the protoplast suspension. 6. Addition of NaN₃ or CCCP resulted in an inhibition of d-xylose uptake. The transport process is tightly coupled to cell metabolism.It is concluded that the metabolic and transport functions of R. gracilis protoplasts equal those of the intact yeast cells. The application of the protoplasts to study some special transport problems revealed: 1. In the course of d-trehalose uptake the disaccharide was cleaved to glucose, which was actually transported across the cell membrane. 2. Densitometry of protoplasts suspensions was found suitable for the continuous recording of sugar uptake processes. This observation is of special importance for further investigations of the oscillations in sugar transport observed earlier (Heller and Höfer, 1973).

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Herrn Professor Dr. Maximilian Steiner zum 70. Geburtstag gewidmet.     

Pentose metabolism byBacteroides ruminicola subsp.brevis strain B14

The fermentation ofd-arabinose byBacteroides ruminicola strain B₁4 occurs in a manner similar to or identical with that shown previously forl-arabinose metabolism by the organism, a combination of hexose resynthesis and the Embden-Meyerhof sequence. The use ofd-arabinose by strain B₁4 was repressed by prior growth in medium containingd-glucose and induced by prior growth in the presence ofl-arabinose ord-xylose. The use ofd-ribose andd-xylose by strain B₁4 is different from that ford-arabinose. During growth in the presence of 1-14C-d-arabinose, labeled acetate, propionate, and succinate were formed, whereas during 1-14C-d-ribose growth only labeled acetate and propionate were obtained. Under the conditions used,d-xylose growth failed to allow formation of acetate, propionate, or succinate. Strain B₁4 incorporates label from 1- or 2-labeled glycine into acetate, propionate, and succinate by a mechanism involving the cleavage of glycine and equilibration of glycine carbons 1 and 2 with different metabolic pools.     

Yeast strains from Livingston Island, Antarctica

Five yeast strains were isolated from soil and moss samples from the Livingston Island (Antarctica) and identified according to morphological, cultural and physiological characteristics. All strains had an optimum growth temperature of 15°C; none grew above 25°C. They assimilatedD-glucose.D-galactose, sucrose, cellobiose, trehalose, 2-keto-d-gluconate,D-xylose,d-ribose and melezitose. Four of them were nonfermentative, only one, which formed pseudomycelium fermented glucose, galactose, trehalose. Two strains were identified as pinkred yeasts belonging to genusRhodotorula—R. minuta andR. mucilaginosa; two were related to the genusCryptococcus—C. albidus andC. laurentii, one wasCandida oleophila.     

The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.