(1,3)-beta-D-Glucan synthase from budding and filamentous cultures of the dimorphic fungus Candida albicans.

UDPglucose:(1,3)-beta-D-glucan 3-beta-D-glucosyltransferase (EC 2.4.1.34) was obtained as a particulate fraction from cell-free extracts prepared after mechanical breakage of cells of a strain of the dimorphic fungus Candida albicans. The properties of this glucosyltransferase were investigated. Budding and filamentous cultures of C. albicans were grown after dilution of a stationary phase inoculum of yeast cells into fresh medium at 30 degrees C and 40 degrees C respectively and the specific activities of (1,3)-beta-D-glucan synthase, obtained from budding and filamentous cultures harvested during the first 3 h of their growth, were compared. 1. UDPglucose was the only glucosyl donor in the reaction (assayed by following the incorporation of radioactivity from UDP[14C]glucose into polymer) and the radioactive product was exclusively beta-(1,3)-glucan. 2. Glycogen synthase activity was not detected. 3. (1,3)-beta-D-Glucan synthase activity was enhanced by ATP and GTP. 4. A threefold increase in the specific activity of the glucosyltransferase was obtained when cell breakage, and subsequent steps in the preparation of the enzyme, were carried out in the presence of 25 microM GTP. A Km value for UDPglucose of 1.5-1.9 mM was obtained for the glucosyltransferase prepared in the presence or absence of GTP. 5. The glucosyltransferase reaction was not affected by ADPglucose, CDPglucose, GDPglucose or glucose 6-phosphate, but was competitively inhibited by TDPglucose. GDPglucose was not a glucosyl donor under the present conditions. 6. There was no evidence that the product was N-glycosidically linked to protein, since the reaction was neither enhanced in the presence of UDP-N-acetylglucosamine, nor inhibited by tunicamycin. 7. The specific activity of the glucosyltransferase from 3-h-old filamentous cultures was about 1.5-times higher than that from 3-h-old budding cultures. 8. The specific activities of (1,3)-beta-D-glucan synthase prepared from budding and filamentous cultures of C. albicans during their first 90 min of growth were similar.

The action of 2-deoxy-D-glucose on the incorporation of glucose into (1----3)-beta-glucan in stationary phase cultures of Candida albicans.

2-Deoxy-D-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1----3)-beta-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-D-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-D-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase. When C. albicans was grown in the presence of 2-deoxy-D-glucose for 24 h, analogue residues became incorporated into the (1----3)-beta-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this beta-glucan fraction was greater than in controls. Pretreatment of cultures with beta-glucanase, or incubation under conditions known to stimulate endogenous beta-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-D-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1----3)-beta-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.