Materials
Protocol
Results
Tips

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1. Harvest log-phase cultures by centrifugation and wash with cold water and TM buffer.
2. Resuspend cells in 1.5 ml TM buffer.  Add equal volume of glass beads (0.45 - 0.55 mm) (Thomas Scientific, Swedesboro, NJ).  Agitate via vortex mixer during six 30 second intervals, between which the samples should be cooled on ice.
3. Recover cell slurry by washing the glass bead mixture 6 times with TM buffer (total 6 ml).  Centrifuge the pooled washings at 3,500 G for 5 min.   Collect the supernatant (wall free extract).
4. Centrifuge the wall-free extracts at 60,000 G for 45 min at 4°C.  Remove the pellet (a mixture of membrane protein), resuspending and homogenizing it in TM buffer (1 ml) containing 33% glycerol for immediate use (or storage at -70°C).
5. Mix 30 µg of membrane protein with 40 µl of the reaction mixture (30 µl of 0.5 M Tris-HCl, pH 7.5, 3 µl of 40 mM MgAc, 2 µl of 0.8 M N-acetylglucosamine (Sigma), 5 µl of 10 mM UDP-N-acetyl-D-[U-¹⁴C] glucosamine (Amersham, Arlington Heights, IL, specific activity: 271 mCi/mmol) and 10 mM UDP-N-acetylglucosamine (sigma)).  For trypsin-activated chitin synthase activity measurements, trypsin (2 µl of 1 mg/ml) from bovine pancreas (type III; Sigma) is added to the membrane preparations that are then incubated at 30°C for 15 min.  Soybean trypsin inhibitor (Sigma) (2 µl of 1.5 mg/ml) is added to terminate trypsin digestion.   Incubate the mixture at 30°C for 30 min.  Stop the reactions by adding 10% (wt/vol) TCA (1 ml). 
6. Collect the chitin precipitate by filtration on 25 mm glass fiber filters (type A/E) (Gelman Science, Ann Arbor, MI). 
7. Wash the filters with 95% ethanol (5 ml) and measure the radioactivity in a liquid scintillation counter.
8. Differences in chitin and chitin synthase activities among groups can be evaluated for statistical significance by the parametric one-way ANOVA Newman-Keuls test for paired data.

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1. The first portion of the Chitin Synthase Activity Assay involves isolating the membrane from the Wd samples.  Steps 1-4 of this protocol essentially replicate the Membrane isolation from Wangiella dermatitidis. Protocol found also at this site. 
2. The amount of membrane protein required for the assay was determined by measuring concentrations of the membrane protein produced using the Commassie Protein Assay Reagent (PIERCE, Rockford, IL).

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This page updated on:
Tuesday, March 04, 2003 12:39:05 AM