Purification and some properties of human erythrocyte calpastatin.
Calpastatin, an endogenous inhibitor protein specifically acting on calpain [EC 3.4.22.17; Ca2+-dependent cysteine proteinase], was purified to apparent homogeneity from the cytosol fraction of human erythrocytes. The yield was 0.38 mg from 400 ml of blood. The purification procedures included DEAE-cellulose and Ultrogel AcA34 gel chromatographies followed by heat treatment and a final gel chromatography on Sephacryl S-200, from which calpastatin was eluted at a position corresponding to a 280,000-dalton molecular mass. The heat treatment at 100 C for 15 min at pH 7.5 very effectively removed contaminant proteins. The homogeneity of the final product was demonstrated on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, giving a single protein band with a calculated molecular weight of 70,000. After re-extraction from the gel, the 70,000-dalton protein readily reassociated to give a 280,000-dalton peak upon Sephacryl S-200 chromatography with a recovery of total inhibitory activity of 42%. Purified calpastatin had an isoelectric point at pH 4.55. It had glutamine as the amino-terminal residue. Amino acid analyses revealed that it contained relatively large amounts of proline, glutamic acid, and lysine, smaller amounts of aromatic amino acids, notably no tryptophan, and no amino sugars. The content of half-cystine was less than one per 643 amino acid residues. These features are in general agreement with those of the reported amino acid composition of Ca2+-protease inhibitor from chicken skeletal muscle [Ishiura et al. (1982) Biochim. Biophys. Acta 701, 216-223], although these inhibitors were found to be definitely different in several other respects. Human erythrocyte calpastatin could inhibit not only calpain of the same origin but also calpains having low and high Ca2+-sensitivity from rat liver.
D000596:Amino Acids / Q000032:analysis; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000097:blood*; D055598:Chemical Phenomena; D002621:Chemistry; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007525:Isoelectric Focusing
D000596:Amino Acids; D002135:Calcium-Binding Proteins; C033668:calpastatin