Isolation and partial characterization of secretory granule membranes from the rat parotid gland

Secretory granule fractions prepared by the Urografin gradient method of Kirshner et al. ( Analyt. Biochem. 52, 589–594) demonstrate an impressive degree of purity as judged by electron-microscopic appearance and chemical and enzymic analyses. Granule membrane preparations, obtained by hypotonic lysis of the secretory granules followed by simple sedimentation of the particulate material, were seen to be significantly contaminated by mitochondria, unlysed granules and other debris. In an effort to improve the purity of the membranes, lysed granules were subjected to discontinuous sucrose-density centrifugation, resulting in the recovery of 3 fractions, one of which contained membrane fragments, free of contamination by other organelles and debris. This membrane fraction, as well as the other two fractions, were characterized with respect to ultrastructural appearance, amino-acid composition and presence of loosely associated proline-rich proteins. The results indicate that previously reported characteristics of rat parotid secretory granule membranes are inaccurate due to the organelle contamination demonstrated in this study.