CHITIN BIOSYNTHESIS AND ULTRASTRUCTURE OF CELLULIN GRANULES IN THE LEPTOMITACEAE

Leptomitacean fungi are unique chitin-synthesizing systems in that they contain chitin in two locations, cell walls and cellulin granules within hyphal vacuoles. These chitin-glucan granules are not known to occur in any other organisms, except for these unusal oomycetes. Electron microscopy has been employed to study the characteristics of cellulin granules in the three genera comprising the Leptomitaceae, and to determine whether granule ultrastructure is similar throughout the family. Results show that cellulin granules are easily recognizable inclusions with the same basic ultrastructural appearance in all three genera. However, there are differences among the genera, with respect to electron-dense structures associated with some of the granules. Electron-dense structures, resembling vesicles, are scattered throughout the matrix of Apodachlya granules, while discrete electron-dense particles surround Apodachlyella granules. Leptomitus granules have no distinctive ultrastructural features.; To study chitin biosynthesis in the Leptomitaceae, the activity and properties of the enzyme that catalyzes chitin synthesis, chitin synthase, have been investigated. Chitin synthase activity has been detected in the three genera comprising the Leptomitaceae. Enzyme activity is inhibited by polyoxin D, and in crude preparations, activity is increased by trypsin. Chitin synthase has been localized in the mixed membrane fraction (74,000Xg pellet) of Apodachlya sp. homogenate. Enzyme activity is "solubilized" from this particulate preparation by digitonin treatment. Properties of chitin synthase are similar in particulate and "soluble" preparations, with respect to optimum pH and temperature, divalent cation requirements, and effect of N-acetyl-D-glucosamine. However, specific activity is much higher in the "soluble" preparation. In many respects, leptomitacean chitin synthase is similar to that in other fungi. However, trysin does not activate chitin synthase in "soluble" preparations of Apodachlya sp. as it does in other investigated fungi. Also, three-to twelve-fold more activity is unmasked by digitonin treatment of Apodachlya sp. preparations than in other fungal preparations.