Abstract
Plasma membranes from rat myometrium preincu- bated in Krebs-Ringer solution and homogenized in a Polytron were isolated by differential and sucrose den- sity gradient centrifugation. The enzyme activities of isolated plasma membranes were analyzed to deter- mine the contamination by mitochondrial inner and outer membranes, endoplasmic reticulum, and lyso- somes. The activities of succinate-cytochrome c reduc- tase and rotenone-sensitive NADH-cytochrome c re- ductase, markers for mitochondrial inner membrane, were enriched in the mitochondrial fraction and de- creased to a very low level in the plasma membrane fraction. Activities of monoamine oxidase and rote- none-insensitive NADH-cytochrome c reductase, sup- posed markers for mitochondrial outer membrane, al- though 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. Cal- cium 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 cou- pling and relaxation in intact muscle. We have reported a standard method for the isolation of subcellular membranes from myometrium and other smooth muscle (1, 2). The fraction markedly enriched in plasma membrane was capable of binding and transporting calcium in the presence of ATP under conditions suggesting that this membrane was involved in the regulation of intracellular * This work was supported by the Medical Research Council
Published Version
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