Sorbose counterflow as a measure of intracellular glucose in baker's yeast.
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Wilkins, Peter O. (New Jersey College of Medicine and Dentistry, Jersey City), and Vincent P. Cirillo. Sorbose counterflow as a measure of intracellular glucose in baker's yeast. J. Bacteriol. 90:1605-1610. 1965.-The intracellular concentration of glucose in metabolizing baker's yeast was determined indirectly from the glucose-induced counterflow of previously accumulated sorbose. The method is based on the concept that sugar transport in yeast is a symmetrical facilitated diffusion. The intracellular glucose concentration increased with an increase in the extracellular concentration and was higher in aerobiosis than in anaerobiosis. The concentrations were considerably greater than those obtained by direct analysis of intracellular glucose. Calculation of the apparent maximal velocity of glucose transport yielded values which varied with the rate of metabolism and the extracellular concentration. This suggests that during glucose metabolism the transport of hexoses includes elements that are not revealed by experiments involving metabolic inhibitors or nonmetabolizable sugars. (+info)
D-arabinose countertransport in Bakers' yeast.
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The initial rate of the glucose-induced countertransport of d-arabinose was measured at several concentrations of extracellular glucose. These data permit the calculation of the intracellular concentration of free glucose, and, if the rate of glucose metabolism is known, the maximal rate of glucose transport can be estimated. Since the maximal transport rate remained essentially constant when the extracellular glucose concentration was increased from 2 to 100 mm, the results are consistent with the hypothesis that, during glucose metabolism, glucose is transported across the yeast cell membrane by a symmetrical carrier system which functions independently of metabolism. (+info)
L-Sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose.
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L-Sorbose degradation in Klebsiella pneumoniae was shown to follow the pathway L-sorbose leads to L-sorbose-1-phosphate leads to D-glucitol-6-phosphate leads to D-fructose-6-phosphate. Transport and phosphorylation of L-sorbose was catalyzed by membrane-bound enzyme IIsor of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system, specific for and regulated by this ketose and different from all other enzymes II described thus far. Two soluble enzymes, an L-sorbose-1-phosphate reductase and a D-glucitol-6-phosphate dehydrogenase, were involved in the conversion of L-sorbose-1-phosphate to D-fructose-6-phosphate. This dehydrogenase was temperature sensitive, preventing growth of wild-type strains of K. pneumoniae at temperatures above 35 degrees C in the presence of L-sorbose. The enzyme was distinct from a second D-glucitol-6-phosphate dehydrogenase involved in the metabolism of D-glucitol. The sor genes were transferred from the chromosome of nonmotile strains of K. pneumoniae by means of a new R'sor+ plasmid to motile strains of Escherichia coli K-12. Such derivatives not only showed the temperature-sensitive Sor+ phenotype characteristic for K. pneumoniae or Sor+ wild-type strains of E. coli, but also reacted positively to sorbose in chemotaxis tests. (+info)
Genes for l-sorbose utilization in Escherichia coli.
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Amongst forty wild strains of Escherichia coli, nine used L-sorbose as a source of carbon and energy and two mutated to use it. Laboratory strains K12, B and C were L-sorbose-negative. Genes for L-sorbose utilization (sor+) were transferred to K12 from six wild strains; genes conferring the mutable phenotype were also transferred. All were cotransducible with metA at 90 min on the linkage map. The most probable gene order was met ace sor pgi mal. Complementation tests identified two genes for L-sorbose utilization. Genetical evidence showed that the catabolite repressor protein of K12 exerted positive control over sor+ genes introduced into K12. The genes for phosphofructokinase (pfkA), the phosphocarrier protein (ptsH) and phosphotransferase enzyme I (ptsI) were required for utilization of L-sorbose. The frequency of transduction of sor+ was low when selection was made for sor+, because L-sorbose partially inhibited the growth of both L-sorbose-negative strains and K12 (sor+) strains. Uridine, thymidine and sorbitol each annulled the inhibition of growth and increased the frequency of transduction of sor+. (+info)
Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.
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Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose. (+info)
Chromosomal alterations of Candida albicans are associated with the gain and loss of assimilating functions.
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We have demonstrated that a normal laboratory strain of Candida albicans spontaneously produces mutants which acquire the ability to assimilate certain carbon sources that are not utilized by the parental strain. The examination of mutants acquiring the ability to utilize either sorbose or D-arabinose revealed a few additional phenotypic changes, including the gain and loss of the capacity to assimilate other carbon sources. The change of assimilation patterns resembled the polymorphic variation of assimilation patterns found among different wild-type strains of C. albicans. Most importantly, these sorbose- and D-arabinose-positive mutants were associated with chromosomal rearrangements, with each class of positive mutants having alterations of specific chromosomes. These findings demonstrated for the first time that chromosomal alterations in C. albicans are involved in genetic variation of fundamental functions of this asexual microorganism. (+info)
The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping-Pong mechanism.
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A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl-maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping-Pong Bi Bi mechanism. The values of the kcat/Km(acceptor) proved that the same configurations of free hydroxyl groups with those of D-glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase. The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of kcat/Km for D-xylose were smaller than that for D-glucose. The value of kcat/Km for maltose was about 20-times larger than that for D-glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites. (+info)
Purification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products.
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A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (M(r)) of 322,800 +/- 18,300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient (S20,w = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of M(r) 76,000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-galactose, methyl beta-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-D-glucose, 5-keto-D-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0-6.5 in 0.1 M potassium phosphate, and its activation energy (delta H degree) for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for D-glucose, L-sorbose, D-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM. (+info)