Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells

Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca²⁺-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 mm) increased the rate of leucine, lysine, and histidine release by ∼12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 mm), dithiothreitol (50 mm), and dimethylthiourea (50 mm) decreased the rate of phenylhydrazine-stimulated amino acid release by ∼30-50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mm) and dimethyl sulfoxide (50 mm) were without significant effect. The calcium chelator, EGTA (10 mm), inhibited phenylhydrazine-stimulated proteolysis by ∼30%. Phenylhydrazine (4 mm) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of ∼28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 mm) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca²⁺-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).