Chitin synthetase activity in Candida albicans: subcellular distribution in yeast cells and protoplasts

Abstract

Chitin synthetase activity was detected in a partly zymogenic form in cell-free homogenates obtained from C. albicans yeast cells and protoplasts of the same type of cells. By isopycnic centrifugation on sucrose gradients of the cell-free homogenates two fractions with chitin synthetase activity were obtained: one was associated with the plasma membrane (labelled with [ 3 H]concanavalin A (con A) and buoyant density 1·195 g ml −1 ) in a partly active state, and the second was in the cytoplasm, where the enzyme was in a particulate fully zymogenic form, lacking affinity to con A. The buoyant density of the enzyme found in this location depended on the method of cell breakage. Lysis of partly regenerated protoplasts gave a value of 1·135 g ml −1 , while cells broken with glass beads gave a major peak of activity at a buoyant density of 1·085 g ml −1 and a second minor peak at the buoyant density obtained in protoplasts (1·135 g ml −1 ). Breakage with glass beads resulted in the release of chitin synthetase, mainly zymogenic, from the membrane fraction (MMF) (buoyant density 1·085 g ml −1 ) whereas the cytoplasmic enzyme activity decreased in parallel with an increase in the buoyant density (from 1·135 g ml −1 to 1·165 g ml −1 ). The extent of both events depended on the conditions of cell disruption. Finally a precursor/product relationship between the cytoplasmic and membrane-bound enzymes could not be demonstrated because neither the subcellular distribution nor the activation state of the enzyme was modified following inhibition of protein synthesis.