Figure 1.

Preparation of trypsinized PDE6. A. The time course of trypsin digestion monitored by SDS–PAGE is shown. Purified PDE6 was mixed with trypsin at 100:1 (weight ratio) and incubated at room temperature for 10, 20, 30, 45 and 60 min. Protein without trypsin was included as a control (Lane 0). At 30 min (arrow), a weak band with a smaller molecular weight appeared. B. Size exclusion chromatography profile of PDE6_t (30 min, room temperature). PDE6_t (solid line) eluted at nearly the same position as untreated PDE6 (dashed line). C. PDE6_t activity with cGMP (2 mM) as substrate. Enzyme activities monitored with the pH–sensitive fluorescent dye SNARF–1were positively correlated with increasing fluorescence intensity over time. PDE6_t was generated as follows: Purified PDE6 was first mixed with trypsin at a 100:1 weight ratio for 10 min. Soybean trypsin inhibitor was then added at a 1:10 mass ratio to stop the reaction (dashed line, –○–). Alternatively, PDE6 was mixed with trypsin–agarose at 30 μg protein per 1 μl agarose for 30 min and the reaction was stopped by passing the sample through a filter to remove the trypsin–agarose. Trypsinized peptides then were removed either by gel filtration (dashed line, –◇–) or buffer exchange with a 50 kDa MWCO filter (Millipore) (solid line, –•–). Activities for PDE6_t prepared by all three methods were identical.