In this study we observed the ATPase
In this study, we observed the ATPase activity of RaGroEL at various temperatures and metal ions conditions and the role of GroES in the ATPase activity of RaGroEL. Furthermore, we quantified the expression of groEL genes under different abiotic stresses, including temperature, pH, salt and oxygen stress.
Materials and methods
Discussion In this study, we used sequence alignment and homology modelling to predict the specific amino obatoclax residues in RaGroEL that were responsible for ATP binding. ATP binding is an important step in GroEL function, leading to the conformational changes necessary for folding the substrate proteins . The ATP binding site in GroEL is one of the most conserved regions of the protein . Our results revealed highly conserved sequences of RaGroEL in regions corresponding to the ATP-binding domains. Previously, it was reported that the chaperonin GroEL required ATP hydrolysis to release the bound polypeptides . We analyzed the effect of GroES on the ATPase activity. The ATP hydrolysis ability of GroEL with GroES has a strong temperature dependence and is relevant for bacteria growth . The high ATPase activity of GroEL at high temperatures suggested that a faster rate of ATP hydrolysis allows the chaperonin to fold many denatured proteins more efficiently under stressful conditions. As observed previously, certain cations were effective in supporting the ATPase activity of GroEL in high temperatures . In this study, the role of potassium in supporting the ability of chaperonin to hydrolyse ATP at 50 °C was similar to that of magnesium. Calcium ions did not support ATPase activity. It has previously been demonstrated that both potassium and magnesium are required for GroEL ATPase activity. Potassium has cooperative effects with magnesium on the release of a bound protein from GroEL . Potassium may increase the affinity of GroEL for ATP , while magnesium activates ATP hydrolysis by GroEL since magnesium can stabilize the structure of GroEL . The growth of bacteria is affected by environmental factors such as temperature, oxygen concentration, pH and salt. When these conditions change substantially, the bacteria will quickly produce heat shock proteins containing GroEL to attenuate the injury of cell and improve survivability in the unfavourable environment . In the present study, we expected to find the relationship between the mRNA expression of groEL and different stress conditions. Temperature is associated with the activity of the enzymes in the bacteria. In high-temperature environments, many intracellular enzymes are denatured and inactivated. These enzymes are reactivated by a large number of molecular chaperone proteins, such as GroEL to maintain the original equilibrium state. Several lines of evidence have shown the cooperative functions of chaperones . Interestingly, the mRNA expression of groEL exhibited little change in 42 °C, it suggests 42 °C is not high temperature for RA-CH-1 since the temperature of poultry is about 42 °C. Under aerobic conditions, the cells are attacked by the atomic oxygen radical anion causing cellular ageing and death. In this study, the change of groEL expression in R. anatipestifer was further improved after prolonged exposure. R. anatipestifer tolerates long periods of oxygen stress and seems to have developed non-transient responses. Under oxidative stress, certain antioxidases, including superoxide dismutase (SOD) and catalase, are involved in scavenging reactive oxygen species (ROC). Methionine sulfoxide reductase apparently serves to protect catalase directly as well as indirectly via the repair of the recovery of GroEL system activity . The GroEL system can also significantly enhance SOD activity recovery and increased the yield of the active enzyme . Moreover, GroEL is closely related to the protective pH shock proteins during pH challenge . The molecular chaperone GroEL helps these proteins to fold into the correct three-dimensional form after their production. In most bacteria, GroEL, as a heat shock protein, is essential in the response to environmental stimulus. In future work, we will investigate whether groEL is also an essential gene in R. anatipestifer.