Purification and characterization of plasma membrane calcium atpase (PMCA) of pig erythrocyte using plasma membrane calcium atpase inhibitor (PMCAI)-sepharose affinity chromatography

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Purification and characterization of plasma membrane calcium atpase (PMCA) of pig erythrocyte using plasma membrane calcium atpase inhibitor (PMCAI)-sepharose affinity chromatography

 

Author: Man, David Shu-ki
Title: Purification and characterization of plasma membrane calcium atpase (PMCA) of pig erythrocyte using plasma membrane calcium atpase inhibitor (PMCAI)-sepharose affinity chromatography
Degree: M.Sc.
Year: 1997
Subject: Calcium in the body
Biological transport
Cell membranes
Hong Kong Polytechnic University -- Dissertations
Department: Multi-disciplinary Studies
Pages: xiii, 66 leaves : ill. ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1421195
URI: http://theses.lib.polyu.edu.hk/handle/200/2347
Abstract: The plasma membrane Ca2+-ATPase inhibitor (PMCAI) from pig erythrocyte was purified to apparent homogeneity using methods involving ultrafiltration, DEAE-Sephadex ion exchange chromatography, and plasma membrane Ca2+-ATPase-Sepharose affinity chromatography. The purified protein revealed two bands using silver staining with apparent molecular mass of 33.5 kDa and 17.6 kDa. To further demonstrate that the PMCAI can directly interact with plasma membrane Ca2+-ATPase (PMCA), affinity chromatographic technique for separation of PMCA was developed using PMCAI as ligand. Solubilized membranes were applied to the column. Proteins eluted with EGTA were compared with the proteins eluted from the calmodulin CaM-Sepharose affinity column. Results showed that the eluted proteins from the CaM-Sepharose affinity column revealed one major band of apparent molecular mass of 80 kDa and two minor bands of apparent molecular mass of about 55 kDa and 23 kDa using coomassie blue staining. And the purified proteins from PMCAI-Sepharose affinity column revealed one coomassie blue stained band with apparent molecular mass of about 90 kDa. Two molecular weights results difference could be accepted within the experimental error as usual and they could be tested by Ca2+-ATPase activity assay to indicate that they are both Ca2+-ATPase. The eluted proteins from PMCAI-Sepharose affinity column were characterized by the Ca2+-ATPase activity assay. The protein was shown to be Ca2+-ATPase that could be stimulated by 0.1% asolectin and/or 10弮g calmodulin.

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