Protein composition of purified chitosomes of Mucor rouxii

Abstract

Different centrifugation conditions were compared for purification of chitosomes (microvesicles containing chitin synthetase). Isopycnic centrifugation of crude chitosome samples from yeast cells of Mucor rouxii on sucrose density gradients, in a vertical rotor, yielded chitosomes of higher purity than before. About 90–96% of the chitin synthetase in purified chitosomes was zymogenic. We estimated that the chitosome population of the yeast form of M. rouxii comprises a miniscule portion (0.17%) of the total cell protein. The polypeptide composition of purified chitosomes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Prominent polypeptide bands were found at 16, 18, 28, 30, 32, 44, 47, and 55 kDa. The 55-kDa polypeptide was most conspicuous. There were also minor bands at 25, 26, 42, 60, 67, and 80 kDa. Our findings show that highly purified populations of chitosomes consist of microvesicles with a characteristic size range, buoyant density, and a complex polypeptide composition.