W-1 solubilization and kinetics of inhibition by cilofungin of Candida albicans (1,3)-beta-D-glucan synthase

Abstract

(1,3)-pi-D-Glucan synthase of Candida albicans was rendered soluble by treatment of membrane preparations with the polyoxyethylene ether detergent W-1. Extraction with 0.025% W-1 at 4°C for 24 h effectively solubilized and activated the enzyme. Under these conditions, >85% of the protein in membrane preparations was released, and about 64% of the glucan synthase activity could be recovered in the soluble form. Soluble enzyme activity was stable for more than 12 days at 4°C. Also, glucan synthase activity in the extracted membrane preparations could be activated to achieve more than twice the enzyme activity in the original, unextracted membrane preparations. The soluble glucan synthase had characteristics similar to those of the membrane-bound enzyme. Soluble glucan synthase had an apparent Km of 2.0 mM, and particulate glucan synthase had an apparent Km of 2.5 mM. Kinetics of cilofungin inhibition for both enzyme preparations were noncompetitive, with an apparent Ki of 2.5 ,uM; both preparations could be inhibited by cilofungin but not by its peptide nucleus or side chain, either alone or in combination. The reaction products from both forms of the enzyme were sensitive to (1,3)-p-D-glucanase degradation but not to a-amylase, a-glucosidase, or proteinase K degradation and thus were shown to be 0(1-*3) glucan.

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

“…A similar response was also reported when the enzyme was extracted with W-1 (Tang & Parr, 1991). However, the addition of Brij-35 during the breakage of cells further extended the half-life.…”

“…Although W-1 has been reported to solubilize glucan synthase from C. albicans membranes, we could not solubilize the enzyme from the microsomal membranes using either W-1 or Brij-35 (data not shown). However, Brij-35 improved the stability and activity of the enzyme in a similar manner to that reported with W-1 treated membranes (Tang & Parr, 1991).…”

“…BSA could also slow down proteolysis and detergents could inhibit proteases. Several detergents have been found to activate fungal glucan synthase (Beauvais e t a/., 1993;Frost e t al., 1992;Quigley e t al., 1988;San-Blas & San-Blas, 1982;Tang & Parr, 1991). Detergents could alter enzyme conformation or membrane fluidity, or act by permeabilizing the membrane to enable better accessibility of substrate and effectors.…”

“…cerevisiae (Frost et al, 1992). Recencly, the detergent W-1 was reported to activate the C. albiians enzyme (Tang & Parr, 1991). NaF and ATP have also been added to in vitro assays as activators (Shematek eta/., 1980;Orlean & Ward, 1983).…”

Compounds active against cell walls of medically important fungi

“…It was determined that isolated chitin synthetase from C. albicans was unaffected by cilofungin, echinocandin B, or papulacandin B. In the first report utilizing a solubilized glucan synthetase, Tang and Parr determined the enzyme kinetics for the enzyme from C. albicans and the inhibition constant for cilofungin (243). The Km of 2.0 mM was similar to that reported earlier for the membrane-bound form (216), and a Ki of 2.5 ,uM was identical to that found by Taft et al (241).…”