Characterization of membrane fractions and isolation of purified plasma membranes from rat myometrium.

Plasma membranes from rat myometrium preincubated in Krebs-Ringer solution and homogenized in a Polytron were isolated by differential and sucrose density gradient centrifugation. The enzyme activities of isolated plasma membranes were analyzed to determine the contamination by mitochondrial inner and outer membranes, endoplasmic reticulum, and lysosomes. The activities of succinate-cytochrome c reductase and rotenone-sensitive NADH-cytochrome c reductase, markers for mitochondrial inner membrane, were enriched in the mitochondrial fraction and decreased to a very low level in the plasma membrane fraction. Activities of monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase, supposed markers for mitochondrial outer membrane, although enriched in the mitochondrial fraction, were also present in the plasma membrane fraction. RNA, protein synthesis, and NADPH-cytochrome c reductase activity, markers for endoplasmic reticulum, were found in small amounts in plasma membrane but in increased amounts in heavier fractions in the sucrose density gradient. The activities of lysosomal enzymes, /3-glucuronidase and /I-galactosidase, were very low in this tissue and present predominantly in mitochondrial and soluble fractions. The plasma membrane fraction was highly enriched with 5’-nucleotidase activity and with lectin and specific oxytocin binding sites. Electron microscopy of the plasma membrane fraction revealed sealed vesicles with little contamination with other membranes. On the basis of protein concentration and marker enzyme activities, it was calculated that this fraction contained 70 to 80% plasma membrane. Calcium uptake by the plasma membrane fraction was the highest among all fractions both in the absence and in the presence of ATP, supporting its strong involvement in calcium exchange during excitation-contraction coupling and relaxation in intact muscle.