INTERACTION OF SULFHYDRYL REACTIVE REAGENTS WITH COMPONENTS INVOLVED IN (1,3)-BETA-GLUCAN SYNTHESIS FROM CANDIDA-ALBICANS

Glucan synthesis was sensitive to several sulfhydryl reacting compound s: mercurials, reversible disulfides, and an alkylating sulfhydryl rea gent( IC50 3-45 mu M). Thiol groups associated with glucan synthesis w ere hydrophilic in nature, since both hydrophilic and hydrophobic reag ents were active. Glucan synthase complex consists of at least two com ponents: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component ( pellet). A rapid separation technique was developed to study sulfhydry l interactions with the complex. The GTP-binding protein was solubiliz ed with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonat e from isolated microsomes of Candida albicans cells grown at either 1 0 or 30 degrees C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of gl ucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost al most 50% activity when preincubated with 2.5 mu M N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catal ytic active site of glucan synthase was not protected with UDP-Glc whe n preincubated with 10 mu M N-ethylmaleimide but the GTP-binding fract ion was partially protected with GTP gamma S.