Characterization of (1,3)-beta-glucan synthase in Candida albicans: microsomal assay from the yeast or mycelial morphological forms and a permeabilized whole-cell assay.
A systematic evaluation of the in vitro (1,3)-beta-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 degrees C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 micrograms protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent Km for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 degrees C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Frost
D J
DJ
Anti-Infective Research Department 47M, Abbott Laboratories, IL 60064-3500.
Brandt
K
K
Capobianco
J
J
Goldman
R
R
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D002176:Candida albicans / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D002463:Cell Membrane Permeability; D003902:Detergents; D054714:Echinocandins; D004795:Enzyme Stability; D005656:Fungal Proteins; D005964:Glucosyltransferases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006160:Guanosine Triphosphate; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D010455:Peptides; D010456:Peptides, Cyclic / Q000494:pharmacology; D000077423:Polidocanol; D011092:Polyethylene Glycols; D029702:Schizosaccharomyces pombe Proteins
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D003902:Detergents; D054714:Echinocandins; D005656:Fungal Proteins; D008565:Membrane Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D029702:Schizosaccharomyces pombe Proteins; D000077423:Polidocanol; D011092:Polyethylene Glycols; C020789:papulacandin B; D006160:Guanosine Triphosphate; C042101:cilofungin; C007572:echinocandin B; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase