Alterations of myosin isoenzymes and ADP/ATP carrier in Goldblatt hypertensive rats (part 3)

Tissues: Fourteen-week-old Goldblatt and control rats were laparotomized under diethylether anesthesia. Blood was collected from the abdominal vein, and the sera were used for biochemical analysis. Hearts were immediately removed and dissected into ventricles. Then they were weighed, frozen in liquid nitrogen and stored at -70°C until use.
Myosin isoenzyme extraction and PGE: Native myosin was extracted from the left ventricles according to the method of Martin et al . All the following procedures were carried out at 2°C. Approximately 0.5 g of minced left ventricular myocardium was homogenized in 40 mmol NaCl, 5 |ig/mL of leupeptin and 3 mmol Na-phosphate buffer (pH 7.0) with a glass homogenizer.The homogenate was centrifuged at 1000 g for 15 mins. The pellet was resuspended in the same solution and centrifuged. The pellet was suspended in 5 mmol dithiothreitol, 5 mmol EGTA, 5 |ig of leupeptin/mL and 0.1 molar Na-pyrophosphate buffer (pH 8.6) for 1 h. The suspension was then centrifuged at 100,000 g for 1 h. The same volume of glycerol was added to the supernatant, which contained the myosin, and the solution was stored at -20°C. Myosin isoenzymes were separated by PAGE in the presence of pyrophosphate according to the method of Hoh et al . The gel contained 3.88% acrylamide and 0.12% N,N’-methylene-bis acrylamide. The running buffer was 20 mmol Na4P2O7 (pH 8.8) in the presence of 10% glycerol. Electrophoresis was carried out for 24 h at 2°C and a constant voltage of 13.3 V/cm. Gels were stained with Coomassie brilliant blue R-250, G-250 and were destained with 7.5% acetic acid and 5% methanol. Bands revealed on the gel were recorded by densitometer. Start online shopping right now and see how great it feels to know you are saving more money than you are spending: all that is possible whenever you at the best pharmacy you can actually choose yourself right now.

This entry was posted in Hypertrophy and tagged ADP/ATP carrier, Goldblatt hypertensive rat, Hypertrophy, Myosin isoenzyme, Pyrophosphate gel electrophoresis.