calpain and phenylhydrazine

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 Ca(2+)-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 approximately 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 approximately 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 approximately 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 approximately 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 f

Topics: Amino Acids; Antibodies; Antioxidants; Blood Proteins; Calpain; Chromatography, High Pressure Liquid; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Erythrocytes; Free Radical Scavengers; Free Radicals; Hemoglobins; Humans; Immunoblotting; Membrane Proteins; Oxygen; Phenylhydrazines

A 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) was found in the cytosol of anemic rat liver. When phenylhydrazine hydrochloride was continuously administered to rats, a 280,000-dalton calpain inhibitor that existed originally in the liver gradually disappeared within two weeks and, concomitantly, a 34,000-dalton inhibitor appeared. The purified 34,000-dalton inhibitor resembles 280,000-dalton inhibitor in that both are heat-stable proteins and do not inhibit papain and trypsin. Unlike the protomers of a 280,000-dalton inhibitor, 34,000-dalton inhibitor does not show any sign of self-association.

Topics: Anemia; Animals; Calpain; Liver; Male; Molecular Weight; Phenylhydrazines; Protease Inhibitors; Rats; Rats, Inbred Strains; Tissue Extracts