Cardiac S-100a 0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies

• Published in: Cell calcium (Edinburgh) Vol. 10; no. 2; pp. 81 - 92
• Main Authors: Donato, R., Giambanco, I., Aisa, M.C., di Geronimo, G., Ceccarelli, P., Rambotti, M.G., Spreca, A.
• Format: Journal Article
• Language: English
• Published: Elsevier India Pvt Ltd 1989
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/0143-4160(89)90048-1

A simple procedure is described for the purification of the αα isoform of S-100 proteins (S-100a 0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0–50%) of the extract; iii) Ca 2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0–0.0 mM CaCl 2 and 0.0–1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a 0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca 2+ for S-100a 0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca 2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a 0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a 0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a 0 behaves as a peripheral membrane protein in cardiac tissue.