Saxitoxin and ouabain binding activity of isolated skeletal muscle membrane as indicators of surface origin and purity

Abstract

A simple biochemical method for identifying and distinguishing transverse tubule and sarcolemma membranes in preparations of skeletal muscle microsomes is proposed and evaluated. This method is based on the previous observation that the ratio of ouabain to saxitoxin binding sites is five-fold higher in the sarcolemma than the transverse tubule. We measured [3H]saxitoxin and [3H]ouabain binding to microsomes of frog, rat and rabbit muscle in the presence of detergents to expose latent sites. A high density fraction (30--40% sucrose) of the membranes was identified as transverse tubule on the basis of a low ouabain/saxitoxin ratio and its association with sarcoplasmic reticulum. A low density fraction (20--30% sucrose) was identified as transverse tubule containing variable amounts of sarcolemma as judged by a higher ratio of ouabain/saxitoxin sites. Our results suggest that this ratio can be used to determine the surface origin of muscle membrane preparations. Several different methods for purifying transverse tubules were compared by this technique.