Isolation and characterization of canine cardiac sarcoplasmic reticulum with improved Ca2+ transport properties.
A procedure has been developed for isolating canine cardiac sarcoplasmic reticulum with considerably improved Ca2+ transport properties and stability. Contamination by mitochondria and sarcolemma is low, and the preparation is at least 85% pure sarcoplasmic reticulum. The preparation exhibits efficient, high activity ATP-dependent, oxalate-facilitated Ca2+ accumulation. At 13-16 microM ionized Ca2+, loading at 37 degrees C is 2.55 +/- 0.08 mumol of Ca2+/mg of protein in 1 min and reaches 9.08 +/- 0.64 mumol of Ca2+/mg of protein. Approximately 1 mol of Ca2+ is transported per mol of ATP hydrolyzed. Ca2+-insensitive ATPase is low. The Ca2+ loading rate and the Ca2+/ATP efficiency are increased by addition of ryanodine. At 1.2 microM ionized Ca2+, where the control rate is significantly higher than values previously reported, the Ca2+ transport rate is further increased 64% by calmodulin, 2.3-fold by cAMP plus cAMP-dependent protein kinase, and 2.5-fold by the combination of these components. The preparation is stable for 24 h at room temperature and for 48 hr at 0 degrees C and can be stored at -70 degrees C with retention of function for more than 1 month. The preparation is further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by phospholipid analysis, and by thin section, freeze-fracture, and negative staining electron microscopy. The analyses indicate that the orientation and turnover number of the cardiac Ca2+ pump protein are similar to those of the skeletal muscle enzyme, and that the major factor in the lower Ca2+ transport rate of cardiac sarcoplasmic reticulum is a lower density of Ca2+ pump polypeptides in the membrane.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D000242:Cyclic AMP / Q000494:pharmacology; D004285:Dogs; D005260:Female; D008297:Male; D008854:Microscopy, Electron; D009206:Myocardium / Q000648:ultrastructure*; D010743:Phospholipids / Q000032:analysis; D011494:Protein Kinases / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000032:analysis / Q000378:metabolism*
D002147:Calmodulin; D010743:Phospholipids; D012433:Ryanodine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium