A novel method for analysing 5′ nucleotidase activity of rainbow trout, Oncorhynchus mykiss, head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5′ nucleotidase activity, and = 105 whole cells or = 104 lysed cells were required. Maximal 5′ nucleotidase activity was found in the range 27-33°C and using a substrate concentration of ≥ 1·5 μmol AMP ml-1 for whole cells and ≥1·5 μmol AMP ml-1 for lysed cells. 5′ Nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing macrophage activating factor (MAF) activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5′ nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.
- 5′ nucleotidase
- Rainbow trout