A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.
Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.
Membrane proteins/enzymes are essential biological components of cells as they play critical roles in various processes1,2,3. Some of the functions may include transport of ions and molecules in and out of the cell/internal compartments (either active/passive), cell-cell recognition, intercellular binding, anchorage/attachment, and sensing of the external environment through integration with the signal transduction machinery under normal and harsh physical and chemical conditions (high salt, low water, high temperature, drug resistance, etc.)3 The....
Access restricted. Please log in or start a trial to view this content.
All animal procedures were performed in accordance with international and local (NORMA Oficial Mexicana NOM-062-ZOO-1999) guidelines for the handling of animals in experimental laboratories51,67,68. Muscle tissue was obtained from wild-type Oryctolagus cuniculus from a local animal handling unit (INE/CITES/DGVS-ZOO-E0055-SLP-98)46. A veterinarian with expertise in laboratory animal management performed the initial muscle dissection and processing. The details of the reagents and the equipment used in this study are listed in the Table ....
Access restricted. Please log in or start a trial to view this content.
SDS-PAGE of SERCA at different stages of purification (Figure 1). The Coomassie blue-stained gel shows the enrichment of the SERCA protein band (apparent molecular weight >100 kDa) as the purification protocol progresses. The protein band corresponding to SERCA shows a purity of >90% after centrifugation on a trehalose concentration gradient. Plot of ATP hydrolysis rate versus ATP concentration (Figure 2A). Michaelis-Menten ATPase kinetic (hyper.......
Access restricted. Please log in or start a trial to view this content.
Most molecules and ions cannot freely cross the cell membranes, e.g., proton (H+) requires a membrane transporter in the plasma membrane of a variety of organisms and organelles such as mitochondria83,84. Cell membranes are selective, and the molecules and ions that cross the cell membranes are diverse, so several types of membrane proteins can be found in the cell, such as (a) ABC transporters, (b) ion channels, (c) membrane-bound ATPases, (d) SLC tra.......
Access restricted. Please log in or start a trial to view this content.
The author declares that he has no competing financial interests.
The author acknowledges the help of Edmundo Mata-Morales in editing the video, VM, Valentín de la Cruz-Torres in purifying the SRVs, Miguel A. Rivera-Moran in SERCA purification and analysis, and Juan C. Gonzalez-Castro, Franco E. Juarez, Alejandra Nevarez, Nicolas Rocha-Vizuet, and Jocelin I. Ramírez-Alonso in video production. No funds, grants, or other support were received to conduct this study.
....Access restricted. Please log in or start a trial to view this content.
Name | Company | Catalog Number | Comments |
ATP | Sigma-Aldrich Corp | A2383 | |
Azolectin from soybean | Sigma-Aldrich Corp | 44924 | |
Benchtop UV transilluminator | Cole-Parmer | EW-97623-08 | Dual intensity High setting is ideal for analytical documentation. Low setting reduces photonicking or photobleaching of gel samples while doing preparative work. |
CaCl2 • 2H2O | Sigma-Aldrich Corp | 223506 | |
Coolpix B500 camera | Nikon Corp | S210 | |
Coomassie brilliant blue G-250 | Bio-Rad | 1610406 | |
Dodecyl maltoside | Sigma-Aldrich Corp | D4641 | |
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid | Sigma-Aldrich Corp | E4378 | |
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate | Sigma-Aldrich Corp | E6511 | |
Fluorescein isothiocyanate | Sigma-Aldrich Corp | F3651 | |
KCl | JT Baker | 7447-40-7 | |
MgCl2 • 6H2O | Sigma-Aldrich Corp | M9272 | |
MOPS | Sigma-Aldrich Corp | M1254 | |
NADH | Chem-Impex International Inc | 230 | |
N-tetradecyl-N,N- dimethyl-3-ammonium-1-propanesulfonate | Sigma-Aldrich Corp | D0431 | |
Phosphoenolpyruvate (PEP) | Chem-Impex International Inc | 9711 | |
Rabbit muscle lactate dehydrogenase | Roche | 10003557103 | |
Rabbit muscle pyruvate kinase | Sigma-Aldrich Corp | P1506 | |
Sodium deoxycholate | Sigma-Aldrich Corp | D6750 | |
Sodium dodecyl sulfate | Bio-Rad | 1610302 | |
Spectropolarimeter | Jasco Corp. | Jasco J1500 | |
Sucrose | Sigma-Aldrich Corp | 84100 | |
Trehalose | Sigma-Aldrich Corp | T0167 | Dihydrate |
Tris(hydroxymethyl)aminomethane hydrochloride | Sigma-Aldrich Corp | 857645 | |
Ultracentrifuge | Beckman | Optima XPN | |
UV/VIS spectrophotometer | Agilent Technologies | 8453 | The Agilent 8453 UV-Vis Spectrophotometer uses a photodiode array for simultaneous measurement of the complete ultra-violet to visible light spectrum |
WiseStir HS- 30E | Daihan Scientific Co. | DH.WOS01010 | Ideal for all disperging and homogenizing applications, designed for tissue grinders. |
Access restricted. Please log in or start a trial to view this content.
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved