Evidence of complex formation between alkaline phosphatase and a pro-apoptotic hemoprotein cytochrome c

Two proteins, alkaline phosphatase (AP) and cytochrome c (cyt c) which seem to be involved in the apoptotic cell death program were examined on their interaction. Intestinal AP affects ferricytochrome c (cyt c(FeIII)) by chan ging its optical properties, redox state and conformation. The effect proce eded over the course of hours with a gradual decrease in free cyt c(FeIII) as the AP concentration increased. A heme containing high molecular species was created in the first stage of interaction of the proteins in neutral, acidic (pH 2.6), alkaline (pH 8.3), low ionic strength (10 mmol/l phosphate ), and high ionic strength (0.5 mol/l NaCl) media. Further complexation was favored by higher pH values and temperature. Differential scanning calorim etry revealed a decrease in enthalpy of the thermodenaturation temperature (T-m) of cyt c at 84.5 degrees C due to the AP addition. Increments of AP i n the mixtures resulted in the appearance of T-m peaks at 68 degrees C and 61 degrees C. Electrophoretic analysis of the commercial samples of intesti nal APs showed main fractions from 63.2 kDa to 72.9 kDa and from 172.9 up t o 179.0 kDa. Changes in positions and intensities of the bands were detecte d upon longer incubation (24 h) with cyt c. The electrophoretic pattern of the bacterial,AP was homogeneous with one fraction of 43.7 kDa showing no a lteration due to the cyt c presence. Gel permeation chromatography of incub ated mixtures of intestinal APs and cyt c confirmed the creation of new hem e containing complexes.