Purification of the Sarco-Endoplasmic Reticulum Ca²⁺-ATPase from Rabbit Muscle

Our research focuses on purifying SERCA, with high structural quality and catalytic activity while investigating whether trehalose stabilizes SERCA during purification, enhancing it's stability and functionality. The determination of the three dimensional structure of P-type ATPases in their oligomeric states. The use of artificial intelligence for ATPases structural modeling, structural dynamics simulations, and molecular docking of ligands to explore disease therapeutics and improve protein function analysis and newer technologies. Current challenges include understanding energy conversion mechanisms in P-ATPases, elucidating how quaternary structure regulates function and activity, and identifying effective inhibitors for therapeutic application in human diseases. We confirm the hexameric state of GS plasma membrane proton-ATPase and demonstrate its structural dynamics dependence or medium viscosity as described by Kramers' theory.

[Narrator] To begin, obtain fast-twitch muscle from wild-type Oryctolagus cuniculus. Suspend it in three volumes of 100 millimolar potassium chloride for maceration. Blend for one minute, followed by one minute of ice-cold rest. Centrifuge the sample at 1,500 g at 4 degrees Celsius for 5 minutes to remove the tissue debris. Collect the supernatant into a tube, then homogenize it with a tissue grinder. Next, centrifuge the homogenate, followed by the supernatant. Suspend the pellets in approximately 40 milliliters of 0.5 molar sucrose. Now, centrifuge the suspension at 12,000 g at 4 degree Celsius for 15 minutes. Dilute the resulting supernatant in 0.6 molar potassium chloride and 0.15 molar sucrose before centrifuging. Suspend the pellet containing sarcoplasmic reticulum vesicle sheets in a buffer containing 0.3 molar sucrose, 0.1 molar potassium chloride, and 5 millimolar Tris-hydrochloride at pH 7.0. Aliquot the sarcoplasmic reticulum vesicles in 1 milliliter volumes. Incubate the sarcoplasmic reticulum vesicle suspension on ice for 10 minutes with gentle agitation, then centrifuge. Suspend the pellet in less than 3 milliliters of 25 millimolar Tris-hydrochloride buffer. After homogenizing the suspension, adjust it to a final volume of 10 milliliters with Tris-hydrochloride buffer. Determine protein concentration using the Lowry assay. Add azolectin at 5 milligrams per milliliters and then add 0.85 Zwittergent 3-14. Homogenize the suspension again and centrifuge at 100,000 g at 4 degrees Celsius for 60 minutes. Collect the supernatant in a new tube. Dilute it in a 1 to 2 ratio with 2 millimolar EGTA at pH 7.2. Pour the suspension gently onto a discontinuous trehalose concentration gradient in Tris buffer. Then centrifuge as before and collect the transparent, slightly yellow pellet. Suspend the pellet gently in a small volume of Tris buffer. Measure the protein concentration, then dilute it to adjust the protein concentration to 2 milligrams per milliliter. For the ATPase activity, prepare 1 milliliter of the reaction mixture. Vortex the reaction assay to homogenize, then incubate it at 37 degrees Celsius for 10 minutes. Add 0.9 units of L-lactate dehydrogenase and 1.5 units of pyruvate kinase. Add 10 micrograms of SERCA to initiate the ATPase reaction. Monitor the absorbance change every second for 600 seconds at 340 nanometers with a spectrophotometer that has a thermostatic cell holder to determine the formation of NAD. For the labeling assay of SERCA with fluorescein isothiocyanate, or FITC, first suspend 20 micrograms of SERCA in 50 microliters of labeling buffer. Vortex to mix well. Then incubate the samples in the dark at room temperature for 15, 10, 7, 5, and 2 minutes. To stop the labeling reaction, add an equal volume of ice-cold stopping buffer. Incubate on ice in the dark for 5 minutes. Subject the FITC-labeled SERCA to SDS-PAGE. Expose the clear gels to ultraviolet light at a wavelength of 302 nanometers and photodocument the result. Finally, stain the gel with Coomassie blue. Photodocument the result. For circular dichroism spectra analysis, first suspend SERCA in 400 microliters of 10 millimolar phosphate buffer of pH 7.0 at 25 degrees Celsius. Load the sample into a cell with path length of 0.1 centimeters. Now, set the far ultraviolet spectra range between 190 and 260 nanometers and the internal resolution and bandwidth to 1 nanometer. Record the circular dichroism signal at a speed of 50 nanometers per minute at 25 degrees Celsius. The Coomassie blue-stained SDS-PAGE gel demonstrated the enrichment of the SERCA protein band as purification progressed. The final SERCA purification step using ultracentrifugation on a trehalose concentration gradient resulted in a purity exceeding 90%. Sarcoplasmic reticulum vesicles contained a high amount of SERCA, with approximately 73% of the total protein in the vesicles corresponding to this P-ATPase. A Michaelis-Menten kinetic pattern was observed for the purified SERCA in the ATP hydrolysis rate versus ATP concentration curve, confirming its enzymatic functionality. The Lineweaver-Burk plot revealed a Michaelis constant of approximately 10 micromoles for ATP, consistent with previously reported values. FITC labeling of SERCA indicated an intact nucleotide binding site. The Coomassie blue-stained SERCA band corresponded exactly to the FITC-labeled band. Circular dichroism spectroscopy confirmed the high structural quality of the purified SERCA, with its spectrum indicating well-defined secondary structure elements.