Influence of growth temperature on structure, thermostability and kinetic properties of the ATPase coupling factor AF1 of a thermophilic blue-green alga (cyanobacterium).
The coupling factor AF1 isolated from cells of the thermophilic blue-green alga Mastigocladus laminosus grown at 40 degrees C, 50 degrees C and 60 degrees C is investigated and compared with the chloroplast coupling factor CF1. It is demonstrated that the structure of AF1 is affected by the growth temperature. The AF1 from 60 degrees C shows a split alpha-band in dodecylsulfate/polyacrylamide gel electrophoresis which is not observed in AF1 from 40 degrees C or from 50 degrees C. Only the AF1 from 60 degrees C aggregates with allophycocyanin. The AF1 from 60 degrees C is stable up to 85 degrees C for 60 min whereas the AF1 from 40 degrees C and 50 degrees C denature between 65 degrees C and 70 degrees C. CF1 is much less stable. In contrast to the structure, the kinetic properties of the coupling factor are not influenced by the growth temperature. Furthermore, the reaction rates of all three AF1 preparations have the same temperature optimum which jis also identical to that of CF1. The kinetic properties of the AF1 from 50 degrees C were determined in more detail. In the absence of ADP the saturation curve for ATP shows Michaelis-Menten kinetics with Km = 480 micromol and V = 30 micromol ATP hydrolyzed X mg protein-1 X min-1. With the addition of ADP this curve becomes sigmoidal and V decreases. This behaviour and the Hill values suggest an allosteric enzyme with two active sites acting cooperatively. ADP as a product of the ATPase reaction inhibits the enzyme. The inhibition is not simply competitive, it also influences the cooperativity of the active sites.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D011487:Protein Conformation; D006180:Proton-Translocating ATPases
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases