A nonradioactive, high throughput assay for chitin synthase activity.
Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.
Lucero
Héctor A
HA
Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University Medical Center, Boston, MA 02118, USA. hlucero@bu.edu
Kuranda
Michael J
MJ
Bulik
Dorota A
DA
D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002686:Chitin / Q000096:biosynthesis / Q000378:metabolism; D002687:Chitin Synthase / Q000235:genetics / Q000378:metabolism*; D003035:Cobalt / Q000494:pharmacology; D003124:Colorimetry; D004229:Dithiothreitol / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009532:Nickel / Q000494:pharmacology; D037121:Plant Lectins; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013025:Glycine max; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D007527:Isoenzymes; D037121:Plant Lectins; D014909:Wheat Germ Agglutinins; D002686:Chitin; D012967:Sodium Dodecyl Sulfate; D003035:Cobalt; D014537:Uridine Diphosphate N-Acetylglucosamine; D009532:Nickel; D006735:Horseradish Peroxidase; D002687:Chitin Synthase; D004229:Dithiothreitol