The effects of ionic concentration and light on the binding of enzymes to rod outer segment membranes.

The binding of cyclic GMP phosphodiesterase (PDE) to bovine rod outer segment membranes has been measured under various conditions of ionic strength, light and darkness and with and without added GTP. Comparison of the effects of magnesium, calcium and sodium ions shows that it is ionic strength rather than the nature of the ion that determines the binding, and that Ca2+ has no special properties in this respect. The level of light-induced activity of the bound PDE has been measured under the different conditions, and this shows that a relatively constant proportion (1-5%) of the bound PDE is in an active site irrespective of how the membranes have been washed. This means that sufficient GTP-binding protein (GBP) is also bound to the membranes to maintain a constant proportion of active PDE. The washed membranes are capable of activating more PDE molecules than those already bound, and even bleached membranes washed twice with GTP-containing, low-ionic strength buffers could activate considerable numbers of added PDE molecules. These experiments suggest that under the ionic strength conditions of the intact cell effectively all of the PDE is membrane bound, and that exposure to light or changes in the Ca2+ concentration are unlikely to cause significant changes in the amount of PDE bound, or to the level of its activity.