Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies.
A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) 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) Ca2+-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 CaCl2 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-100a0/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 Ca2+ for S-100a0 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 Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 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-100a0 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-100a0 behaves as a peripheral membrane protein in cardiac tissue.
Donato
R
R
Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy.
Giambanco
I
I
Aisa
M C
MC
di Geronimo
G
G
Ceccarelli
P
P
Rambotti
M G
MG
Spreca
A
A
D000818:Animals; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002847:Chromatography, Agarose / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009206:Myocardium / Q000032:analysis* / Q000166:cytology / Q000378:metabolism; D009418:S100 Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
D015415:Biomarkers; D002135:Calcium-Binding Proteins; C030564:S-100 calcium-binding protein alpha subunit; D009418:S100 Proteins; D002118:Calcium