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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

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Protocol

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 ....

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Results

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.......

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Discussion

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.......

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Disclosures

The author declares that he has no competing financial interests.

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
ATPSigma-Aldrich CorpA2383
Azolectin from soybeanSigma-Aldrich Corp44924
Benchtop UV transilluminator Cole-ParmerEW-97623-08Dual intensity High setting is ideal for analytical documentation. Low setting reduces photonicking or photobleaching of gel samples while doing preparative work.
CaCl2 • 2H2OSigma-Aldrich Corp223506
Coolpix B500 camera Nikon CorpS210
Coomassie brilliant blue G-250Bio-Rad1610406
Dodecyl maltosideSigma-Aldrich CorpD4641
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acidSigma-Aldrich CorpE4378
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate Sigma-Aldrich CorpE6511
Fluorescein isothiocyanateSigma-Aldrich CorpF3651
KClJT Baker7447-40-7
MgCl2 • 6H2OSigma-Aldrich CorpM9272
MOPSSigma-Aldrich CorpM1254
NADHChem-Impex International Inc230
N-tetradecyl-N,N- dimethyl-3-ammonium-1-propanesulfonateSigma-Aldrich CorpD0431
Phosphoenolpyruvate (PEP)Chem-Impex International Inc9711
Rabbit muscle lactate dehydrogenaseRoche10003557103
Rabbit muscle pyruvate kinaseSigma-Aldrich CorpP1506
Sodium deoxycholateSigma-Aldrich CorpD6750
Sodium dodecyl sulfateBio-Rad1610302
Spectropolarimeter Jasco Corp.Jasco J1500 
SucroseSigma-Aldrich Corp84100
TrehaloseSigma-Aldrich CorpT0167Dihydrate
Tris(hydroxymethyl)aminomethane hydrochlorideSigma-Aldrich Corp857645
UltracentrifugeBeckmanOptima XPN
UV/VIS spectrophotometer Agilent Technologies8453The Agilent 8453 UV-Vis Spectrophotometer uses a photodiode array for simultaneous measurement of the complete ultra-violet to visible light spectrum
WiseStir HS- 30EDaihan Scientific Co.DH.WOS01010Ideal for all disperging and homogenizing applications, designed for tissue grinders.

References

  1. Schuberth, C., Wedlich-Söldner, R. Building a patchwork - The yeast plasma membrane as model to study lateral domain formation. Biochim Biophys Acta Mol Cell Res. 1853 (4), 767-774 (2015).
  2. Douglas, L. M., Konopka, J. B. Fungal membrane organizatio....

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Sarco Endoplasmic Reticulum Ca2 ATPaseSERCAP type ATPasesTrehaloseMembrane Protein PurificationBiochemical CharacterizationCentrifugationEnzyme KineticsSDS PAGECircular Dichroism SpectroscopyFunctional StudiesStabilizing Additive

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