SUMMARY: Chitin synthase has been characterized from the stipes of by a number of techniques. In the absence of digitonin, on gel filtration columns and during electrophoresis the enzyme showed properties consistent with having molecular weights from 1.5 × 10 to several million, suggesting its reversible aggregation into large multimolecular units. Gel chromatography in buffers containing digitonin gave highly reproducible results, and when followed by anion-exchange chromatography gave preparations with very high activity [e.g. 3.4 μmol substrate incorporated min (mg protein)] and apparent molecular weight 8.0 × 10. The best purification (140-fold) was achieved by gel chromatography followed by copper chelate affinity chromatography, giving a nearly pure enzyme preparation of activity 4.7 μmol substrate incorporated min (mg protein), which showed only one band of molecular weight 6.7 × 10 on SDS-polyacrylamide electrophoresis. These purified preparations were free of the nucleoside diphosphatase and protease activities that were present in the early stages of purification.


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