(1,3)‐β‐d‐Glucan Synthase from Budding and Filamentous Cultures of the Dimorphic Fungus Candida albicans

Abstract

UDPglucose: (1,3)‐β‐d‐glucan 3‐β‐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°C and 40°C respectively and the specific activities of (1,3)‐β‐d‐glucan synthase, obtained from budding and filamentous cultures harvested during the first 3 h of their growth, were compared. 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 β‐(1,3)‐glucan. Glycogen synthase activity was not detected. (1,3)‐β‐d‐Glucan synthase activity was enhanced by ATP and GTP. 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 μM 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. 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. 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. 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. The specific activities of (1,3)‐β‐d‐glucan synthase prepared from budding and filamentous cultures of C. albicans during their first 90 min of growth were similar.

Characterization of (1,3)- -glucan synthase in Candida albicans: microsomal assay from the yeast or mycelial morphological forms and a permeabilized whole-cell assay

“…Enzyme characteristics of C. albicans in this study are summarized in Ta1,le 3. Assays conducted using Brij-35 gave temperature and Klnapy values that were comparable with those reported previously for C. albicans (Orlean, 1982).…”

“…As expected, glucan synthase activity was stimulated by either GTP o r ATP in both morphological forms (Orlean, 1982). However, activation by GTP required a n additional factor, either BSA o r a detergent.…”

“…Only one report of glucan synthase activities has appeared using similar assay conditions in the mycelial and yeast phases of C. albicans but a permeabilized system has not been reported (Orlean, 1982). In order to evaluate antifungal agents we needed a uniform assay system that could be used not only with isolated membranes, but also with a whole-cell system.…”

“…However, activation by GTP required a n additional factor, either BSA o r a detergent. BSA (0-8 %) is added typically to assays with S. cerevisiae (Shematek e t al., 1980) and C. albicans (Orlean, 1982) glucan synthase, while the detergents Brij-35 (0.5%) a n d W-1 (0.025%) have stimulated activities f r o m S. cerevisiae (Frost e t al., 1992) and C. albicans ( T a n g & Parr, 1991), respectively. 4'-1 and with the ATP/BSA treatment activity was only 23 of the GTP/BSA control.…”

“…In addition to GTP, many other effectors must be added to microsomal glucan synthase assays to maximize in vitro catalytic activity. BSA activates the enzyme from Saccharortzyces cerevisiae (Shematek e t al., 1980), C. albicans (Orlean, 1982) and from other fungal sources (RuizHerrera, 1992). BSA could be substituted with plactoglobulin in 5'.…”