Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product.

M S Kang,N Elango,E Mattia,J Au-Young,P W Robbins,E Cabib

doi:10.1016/s0021-9258(17)42698-6

Abstract

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.

Highlights

From the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205
We found that chitin synthetase catalyzes a unidiamide gel electrophoresis in the presence of sodium rectional synthesis of chitin in the plasma membrane [7]
Soluble proteins were removed by centrifugationandtheparticulatematerial was extracted with digitonin to solubilize chitin synthetase

Summary

Introduction

In the course of those investigations it was discovered that chitin synthetase, aplasmalemma-bound enzyme, is presentin the cell as a zymogen and can be converted into an active form by partial proteolysis [5, 6]. N-Acetylglucosamine stimulatedthe purified synthetaseonly slightlyand didnot participate as a primer in the amount of synthetase is trapped in the chitin formed in the reaction and can be eluted from it. This is the crucial step in the procedure for isolation of the enzyme that we describe in thisreport. The same chain length, somewhat more than 100 units ofGlcNAc, was determined in samples of EXPERIMENTALPROCEDURES chitin that had been synthesized either in vivo, or with

Methods

Results

Conclusion