A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature.
Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D005614:Freeze Fracturing; D008081:Liposomes; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008121:Nephropidae; D010713:Phosphatidylcholines; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases