Figure 4.

Protease inhibitors prevent the Ca²⁺- or phospholipase D (PLD)- induced proteolysis of [¹⁴C]et-eEF1A at pH 6.8 and pH 7.2. A, Either benzamidine (Ben) alone or a cocktail (Inh) of aprotinin (5 mm), leupeptin (3.5 mm), and benzamidine (100 mm) were added to microsomes (112 μg of membrane protein) for 1 min, followed by the addition of phospholipase D (5 units) or Ca²⁺ (100 μm) as indicated, or microsomes were treated with CaCl₂ (100 μm), MgCl₂ (100 μm), or MnCl₂ (100 μm). The reactions were incubated at 25°C with shaking (200 rpm) for 10 min, as described in “Materials and Methods” and analyzed by TCA precipitation. B, The reaction conditions are the same as in A except the pH of the reactions was 7.2. The recovery of [¹⁴C]et-eEF1A was analyzed by TCA precipitation. The means of four values from two experiments are reported.