Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Summary

This protocol describes the purification of F₁-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Abstract

F₁-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F₁-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F₁-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F₁-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F₁-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F₁-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F₁-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F₁-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F₁-ATPase isolation from other organisms.

Introduction

The F-type ATP synthases are membrane-bound rotating multiprotein complexes that couple proton translocation across energy-transducing membranes of bacteria, mitochondria, and chloroplasts with the formation of ATP. Molecular details of the rotational mechanism of ATP synthesis are known mainly because of structural studies of purified bacterial and mitochondrial ATP synthases and their subcomplexes¹. F-type ATP synthase is organized into membrane-intrinsic and membrane-extrinsic moieties. The membrane-extrinsic part, known as F₁-ATPase, contains three catalytic sites, where the phosphorylation of adenosine diphosphate (ADP) to ATP o....

Protocol

1. Buffers and Solutions

1. Prepare the solutions listed below. Degas all buffers for liquid chromatography. Add ADP, benzamidine, and protease inhibitors just before use.
   1. Prepare buffer A: 50 mM Tris buffer with hydrochloric acid (Tris-HCl) pH 8.0, 0.25 M sucrose, 5 mM benzamidine, 5 mM aminocaproic acid (ACA), and protease inhibitors (10 µM amastatin, 50 µM bestatin, 50 µM pepstatin, 50 µM leupeptin, and 50 µM diprotin A).
   2. Prepare buffer B: 50 mM Tris-HCl pH 8.0,.......

Discussion

The protocol for F₁-ATPase purification from T. brucei was developed based on previously published methods for the isolation of F₁-ATPase complexes from other species^13,14. The method does not require any genetic modification (e.g., tagging) and yields a fully active complex with all subunits present. The crucial step is the chloroform-facilitated release of the F₁-ATPase from the membrane-attached part of the en.......

Materials

Name	Company	Catalog Number	Comments
Chemicals
Adenosin Diphosphate Disodium Salt (ADP)	Applichem	A0948
Amastatin Hydrochloride	Glantham Life Sciences	GA1330
Aminocaproic Acid	Applichem	A2266
BCA Protein Assay Kit	ThermoFischer Scientific/Pierce	23225
Benzamidine Hydrochloride	Calbiochem	199001
Bestatin Hydrochloride	Sigma Aldrich/Merck	B8385
Chloroform	Any supplier
cOmplete Tablets, Mini EDTA-free	Roche	4693159001	Protease inhibitor cocktail tablets
Ethylenediaminetetraacetic Acid (EDTA)	Any supplier
Hydrochloric Acid	Any supplier		For pH adjustment
Ile-Pro-Ile	Sigma Aldrich/Merck	I9759	Alias Diprotin A
Leupeptin	Sigma Aldrich/Merck	L2884
Magnesium Sulfate Heptahydrate	Any supplier
Pepstatin A	Sigma Aldrich/Merck	P5318
Protein Electrophoresis System	Any supplier
Sodium Chloride	Any supplier
Sucrose	Any supplier
Tris	Any supplier
Name	Company	Catalog Number	Comments
Consumables
Centrifuge Tubes for SW60Ti, Polyallomer	Beckman Coulture	328874
DounceTissues Homogenizer 2 mL	Any supplier
Glass Vacuum Filtration Device	Sartorius	516-7017	Degasing solutions for liquid chromatography
HiTrap Q HP, 5 mL	GE Healthcare Life Sciences	17115401	Anion exchange chromatography column
Regenaretad Cellulose Membrane Filters, pore size 0.45 μm, diameter 47 mm	Sartorius	18406--47------N	Degasing solutions for liquid chromatography
Superdex 200 Increase 10/300 GL	GE Healthcare Life Sciences	29091596	Size-exclusion chromatography column
Vivaspin 6 MWCO 100 kDa PES	Sartorius	VS0641
Name	Company	Catalog Number	Comments
Equipment
AKTA Pure 25	GE Healthcare Life Sciences	29018224	Or similar FPLC system
Spectrophotometer Shimadzu UV-1601	Shimadzu		Or similar spectrophotometer with kinetic assay mode
Ultracentrifuge Beckman Optima with SW60Ti Rotor	Beckman Coulture		Or similar ultracentrifuge and rotor
Ultrasonic Homogenizer with Thin Probe, Model 3000	BioLogics	0-127-0001	Or similar ultrasonic homogenizer