In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.
Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.
Taft
C S
CS
Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver 80262.
Zugel
M
M
Selitrennikoff
C P
CP
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology*; D002499:Centrifugation, Density Gradient; D054714:Echinocandins; D005656:Fungal Proteins; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008565:Membrane Proteins; D009492:Neurospora crassa / Q000201:enzymology; D010455:Peptides; D010456:Peptides, Cyclic / Q000494:pharmacology; D011523:Protoplasts / Q000201:enzymology; D029702:Schizosaccharomyces pombe Proteins; D013013:Sorbose / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000494:pharmacology
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D054714:Echinocandins; D005656:Fungal Proteins; D008565:Membrane Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D029702:Schizosaccharomyces pombe Proteins; D014530:Uridine Diphosphate; C011634:aculeacin A; C020789:papulacandin B; C042101:cilofungin; C007572:echinocandin B; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D013013:Sorbose