ABSTRACIMembranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.A band ofnitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton tramport into membrane vesicles, as measured by the quenching of florescence of acridine orange in the presence of ATP and Mg2@. The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by C1-, but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N'-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar N03-; but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by C17, inhibited by NO03, and showed a high pH optimum and a substrate specificity similar to the ATase, incling some proton transport driven by GTP and UTP.The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H' transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H+-pumping ATPase of red beets originated from the tonoplast. Tonoplast Ht-ATPases with similar properties appear to be widely distributed in higher plants and fgi.
The substrate-binding site of endo-I ,4-/?-xylanase of the yeast Cryptococcus albidus was investigated using, 1,4-/?-xylooligosaccharides (1 -3H)-labelled at the reducing end. Evaluation of the affinities of ten imaginary subsites by the method of Suganuma et al. [1978, J. Biochem. (Tokyo) 84, pointed out that the substrate-binding site of the enzyme is composed of four subsites and that the catalytic groups are localized in the centre. The imaginary subsites on the left-hand side of the binding site ('non-reducing-end' side) showed little or no affinity to bind xylosyl residues. For the subsites on the right-hand side of the binding site ('reducingend' side) negative values of affinity were obtained, which means this region of the enzyme is unfavourable for complexing with xylosyl residues. As a consequence of the asymmetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference for attacking near the reducing end of the substrate. Regardless of the length of [l-3H]xylooligosaccharides, [l-3H]xylobiose was the prevailing reaction product at an early stage of hydrolysis, and frequency distribution of bond cleavage decreased from the second glycosidic bond towards the non-reducing end.Additional information on the substrate-binding site of C. albidus /?-xylanase was obtained by evaluating the efficiency of xylose, xylobiose, methyl P-D-xyloside and pknyl P-D-xyloside to serve as glycosyl acceptors in the transglycosylic reactions proceeding at high concentrations of xylotriose.Studies on the action pattern of polysaccharide hydrolases using the corresponding oligosaccharide substrates led to a concept that the enzyme attacking a polysaccharide chain interacts with the substrate through several glycosyl units at the same time [l -71 and that the interaction of a given part of the enzyme with a single glycosyl unit is more or less independent on the rest of the substrate molecule [8 -121. The binding site of an enzyme is thus considered for an array of subsites, each of which is capable of binding one sugar unit. Catalytic groups are localized between two subsites, and their localization and the number of subsites is a characteristic feature of a polysaccharides hydrolase [4 -121.The present literature offers two methods, similar in principle, for determination of the number of subsites in polysaccharide hydrolases leading simultaneously to localization of the catalytic groups [I 3,141. Both methods are based on product analysis of terminally labelled oligosaccharides under conditions of unimolecular hydrolysis, i.e. when bisubstrate processes such as termolecular shifted binding or transglycosylation reactions are eliminated. Both methods also require some kinetic data for oligosaccharide. Perhaps as a coincidence, multimolecular reactions are characteristic for the mechanism of substrate degradation by lysozyme [5,8] and cc-amylases [6,14-161, the active sites of which are well characterized [S, 141. Tn one of the preceding papers [17] we have shown that...
During growth on wood β‐1,4‐xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo‐1,4‐β‐xylanase.The function of the extracellular β‐xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell‐bound β‐xylosidase (xylobiase). Aryl β‐xylosidase activity detected in the culture fluid was shown to be due to low affinity of β‐xylanase for p‐nitrophenyl β‐D‐xylopyranoside. This property of β‐xylanase was preserved after purification of the enzyme by chromatography on DEAE‐cellulose, CM‐Sephadex and Biogel A 1.5 m or Biogel P 100. Purified β‐xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular β‐xylanase and intracellular β‐xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified β‐xylanase were unable to hydrolyze cellulose or its soluble derivatives.
The mechanism of inhibition by 2-deoxy-~-glucose of the synthesis of yeast wall polysaccharides and glycoproteins was investigated in Saccharomyces cerevisiae cells and protoplasts. The extent of the inhibition of mannan and glucan synthesis was found to be dependent on whether glucose or mannose was used as the carbon source in the medium. During growth on glucose, 2-deoxy-~-glucose inhibited more intensively mannan than glucan formation. Biosynthesis of wall glucan was strongly suppressed in mannose medium. Selective incorporation of 2-deoxy-~-glucose occurred into that polysaccharide, synthesis of which was more inhibited under given conditions. Suggestive evidence has been obtained that the decisive factor for the proportion of glucan and mannan in the walls is the direction of glucose 6-phosphate/mannose 6-phosphate interconversion dependent on the exogeneous hexose.No close correlation was found between the inhibition of mannan synthesis and the appearance of the mannan-protein enzymes invertase and acid phosphatase. Effect of 2-deoxy-~-glucose was therefore investigated on the parallel synthesis of protein, mannan and several extracellular and intracellular enzymes in protoplasts grown on glucose and mannose. The results obtained pointed out that the hindrance of the secretion of mannan-protein enzymes is of a complex nature and related more to the inhibition of synthesis of the protein moiety than to the inhibition of glycosylation. Synthesis of several enzymes was found to be a subject of a metabolic control by 2-deoxy-~-glucose or its metabolites.
The growth inhibition and the lysis of Saccharomyces cerevisiae caused by 2deoxy-D-glucose (2-DG) were shown to be a consequence of unbalanced cellular growth and division. The lysis, but not the repression of growth and osmotic fragility of cells, could be suppressed by the addition of mannitol as an osmotic stabilizer. This result, as well as the morphological changes observed in the cells and changes in the chemical composition of the cell walls, showed that S. cerevisiae grown in the presence of 2-DG formed weakened cell walls responsible for the osmotic fragility. Evidence is presented for the first time demonstrating the incorporation of 2-DG into yeast cell wall material. Other data suggest that the inhibition of yeast growth by 2-DG results from an interference of phosphorylated metabolites of 2-DG with metabolic processes of glucose and mannose involved in the synthesis of structural cell wall polysaccharides.
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