Isolation of a Calcium-Sequestering Protein from Sarcoplasmic Reticulum

Abstract

An acidic protein has been extracted from sarcoplasmic reticulum with KCI and deoxycholate. believe that it is a major binding site for calcium in sarcoplasmic reticulum. We propose that the protein be designated Calsequestrin. MATERIALS AND METHODSPreparation of the calcium-sequestering proteinStep 1. Sarcoplasmic reticulum was prepared from two 14.4-kg albino rabbits and was extracted with deoxycholate as described (8), except that dithiothreitol (1 mM) was present during the extraction and the supernatant, after centrifugation at 165,000 X g, was retained as the source of the binding protein. The deoxycholate extract (about 80 ml) was dialyzed, first for 4 hr and then for 18 hr at 12'C, against 3 liters of 5 mM Tris * HCl, pH 7.5. At 4 and 22 hr, insoluble protein was removed by centrifugation at 165,000 X g for 30 min.Step 2. The supernatant solution (35-to 40-ml aliquot) was passed through a 1.5 X 18 cm column of DEAE-cellulose and the column was eluted with 400 ml of 5 mM Tris -HCl, pH 7.5, that contained a linear gradient of KCl from 0 to 0.7 M. The last peak to be eluted, at a KCl concentration of about 0.48 M, was collected, concentrated by ultrafiltration under pressure (Amicon Diaflo apparatus) to 8 ml, and dialyzed overnight against a solution of 0.15 M KCl-0.01 M Tris -HCl, pH 8.0.Step 3. The dialyzed sample was passed through a 2.5 X 40 cm column of Sephadex G-200 equilibrated with 0.15 M KCl-0.01 AM Tris HCl, pH 8.0. Material absorbing at 280 nm was eluted in a sharp peak at the void volume, in a second symmetrical peak, and in a tail following the second peak. Fractions making up the second symmetrical peak were collected, concentrated by Diaflo filtration to 10 ml, and dialyzed overnight against 10 mM potassium phosphate, pH 7.0.Step 4. The dialyzed sample was applied to a column (7 g) of hydroxylapatite (Biogel HTP) equilibrated with 10 mM potassium phosphate pH 7.0. The column was eluted with 400 ml of a linear gradient of potassium phosphate from 10 to 600 mM. The last peak to be eluted, at a phosphate concentration of about 330 mM, was concentrated to 5 ml and dialyzed overnight against a solution of 5 mM Tris .HC1, pH 7.5. The solution, containing purified protein (about 1 mg per ml), was stored at -20°C. Analytical methodsCalcium transport was measured (10) at a protein concentration of 200 ug/ml. After 10 min at 24°C, 0.5 ml was filtered

Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity

“…In fact, mitochondria, jointly with the ER, are the two main organelles in charge of keeping calcium cell homeostasis. However, while in the ER the levels of free calcium are kept within the physiological range by a group of proteins called calsequestrins (MacLennan & Wong, 1971), mitochondria lack any specific protein to exert this action, and the mechanism governing this process is still unclear.…”

Comparing Skeletal and Cardiac Calsequestrin Structures and Their Calcium Binding

“…Because of this Ca 2ϩ -buffering capacity of CSQ in the lumenal space, the concentration of free Ca 2ϩ in the sarcoplasmic reticulum (SR) can be maintained below the inhibitory level of the Ca 2ϩ pump (1 mM), and simultaneously, the SR can maintain the ability to rapidly deliver a high capacity Ca 2ϩ signal to the cytoplasm. Even though the lumenal space is minuscule compared with the extracellular space, the high concentrations (ϳ100 mg/ml) of CSQ make the SR an efficient storage compartment for Ca 2ϩ (2).…”

Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections.

“…Fragmented sarcoplasmic reticulum has been separated by density gradient centrifugation into heavy and light fractions (3,19) . Both heavy and light fractions contained the Ca" + Mg"-dependent ATPase (Ca 2+ + Mgt+-ATPase), the protein concerned with Ca" uptake (16,17), but only the heavy fraction contained calsequestrin, the protein that probably functions as a luminally located, calcium-sequestering agent (17,18) . Meissner (19) noted a correlation between the presence of matrix material in the heavy fraction and the presence of calsequestrin and proposed that the matrix was composed of calsequestrin .…”