Vacuolar Membranes: Isolation from Yeast Cells

Abstract

α-Mannosidase was found associated with the vacuolar membranes of yeast. The vacuoles were isolated by flotation from osmotically disrupted spheroplasts of Saccharomyces cerevisiae. The enzyme was used as marker for isolating vacuolar membrane fragments directly from whole cells which were mechanically disintegrated. Over 90% of the total α-mannosidase was recovered in the particulate fraction. The enzyme was present in all of the fractions obtained upon differential centrifugation. Density gradient centrifugation in Urografin (5 - 20% w/v) of preparations obtained by differential centrifugation between 20 000 and 50 000 × g did not result in density equilibrium of the membrane. An isolation procedure involving a sedimentation velocity cut in Urografin gradients has, therefore, been worked out.