The Influence Of The Microdomain Lipid Rafts On Ca～(2+)-ATPase In The Cardiac Sarco(endo)plasmic Reticulum

The sarco (endo) plasmic reticulum Ca²⁺-ATPase (SERCA) and Plasma membrane Ca²⁺-ATPase (PMCA) is a very important ATPase which functions to reduce the intracellular free Ca²⁺ through pumping excess Ca²⁺ from cytoplasm into sarcoplasmic reticulum (SR). My thesis focuses on the regulation of SERCA and PMCA by lipid rafts. It consists of three parts:First, we prepared SR vesicles from porcine cardiac by differential centrifugation and extraction with solution of high ionic strength, and then the vesicle wre detected morphologically, characteristically and functionally to be sealed, and relatively devoid of marker enzymea for plasma membranes and mitochondria. The SR had the activity of ATP hydrolysis and calcium uptake, and which could be completely inhibited by the specific inhibitor of SERCA ---- thapsigargin. These results indicated the highly purified SR vesicles were obtained.Second, Lipid rafts are not only restricted to the plasma membranes, but indications for the existence in the Golgi complexes, and endoplasmic reticulum (ER) membranes along the secretory pathway have been published. SR is the specific form of ER in the myocytes, which functions at maintaining the intracellular Ca²⁺ homeostasis. We characterized detergent-resistant membranes (DRM) from SR membranes isolated by using 1% Triton X-100 and sucrose density gradient flotation. These SR-derived detergent-insoluble complexes (SRICs) had a low buoyant density and were highly enriched in sphingomyelin and cholesterol. Marker glycolipid of DRM, such as GM1 was also concentrated in SRICs. Caveolin-3 is the structural and charscteristic protein of caveolae, and located on the sarcolemma. The results of western blot showed the concentration of Cav-3 in SRICs. These results suggest that "lipid rafts" may exist in SR membranes.