A high throughput in vitro assay for fungal (1,3)beta-glucan synthase inhibitors

J Antibiot (Tokyo). 1994 Sep;47(9):1001-9. doi: 10.7164/antibiotics.47.1001.

Abstract

An in vitro assay for (1,3)beta-glucan synthase activity from the filamentous ascomycete Neurospora crassa, suitable for use as a high throughput screen for enzyme inhibitors is described. Samples were added to 25 microliters reaction mixtures in 96-well V-bottom microtiter plates and plates incubated at 22 degrees C. Reactions were terminated by the addition of TCA and the contents transferred to a Milliblot D apparatus containing Inotech 201-A glass fiber filters. Filters were washed to remove unincorporated substrate and the amount of 14C-labeled (1,3)beta-glucan formed by each reaction was quantitated using a Phosphorlmager. As little as 50 ng of an inhibitor with a Ki of 4 microM was detected by this assay. This assay is rapid and cost effective, permitting its use as a screen to detect compounds that inhibit (1,3)beta-glucan synthase activity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antifungal Agents / analysis
  • Filtration
  • Glass
  • Glucans / biosynthesis
  • Glucosyltransferases / antagonists & inhibitors*
  • Kinetics
  • Membrane Proteins*
  • Neurospora crassa / enzymology*
  • Schizosaccharomyces pombe Proteins*
  • Uridine Diphosphate Glucose / metabolism
  • beta-Glucans*

Substances

  • Antifungal Agents
  • Glucans
  • Membrane Proteins
  • Schizosaccharomyces pombe Proteins
  • beta-Glucans
  • beta-1,3-glucan
  • Glucosyltransferases
  • 1,3-beta-glucan synthase
  • Uridine Diphosphate Glucose