Canine traceealis membrane fractionation and characterization

Canine trachealis was homogenized and the various membrane fractions isolated by differential centrifugation and discontinuous sucrose gradient centrifugation. A membrane fraction enriched in the plasma membrane marker enzymes 5′-nucleotidase (5-fold) and K+-activated ouabain sensitive p-nitrophenylphosphatase (3-fold) was obtained. The fraction contained very low levels of the inner mitochondrial marker succinate: cytochrome c oxidoreductase. These plasma membrane vesicles showed higher ATP-dependent Ca-uptake (20 μmoles/g protein) than any other submicrosomal fraction. The active Ca-uptake was enhanced by oxalate. The Ca taken up by the plasma membrane vesicles was released instantaneously by dilution in 5mM EGTA and 10μM A23187 and more slowly by dilution only in 5mM EGTA.