Name: Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei (Video) | JoVE Journal
Uploaded: 2019-01-22T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Learn to purify F1-ATPase from Trypanosoma brucei using classical biochemical methods. This protocol yields highly pure enzyme suitable for structural studies and drug discovery applications.

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5 minutes

This work was funded by the Ministry of Education ERC CZ grant LL1205, the Grant Agency of Czech Republic grant 18-17529S, and by ERDF/ESF project Centre for research of pathogenicity and virulence of parasites (No. CZ.02.1.01/0.0/0.0/16_019/0000759).

1. Buffers and Solutions
<ol>
	<li>Prepare the solutions listed below. Degas all buffers for liquid chromatography. Add ADP, benzamidine, and protease inhibitors just before use.
	<ol>
		<li>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 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A).</li>
		<li>Prepare buffer B: 50 mM Tris-HCl pH 8.0, 0.25 M sucrose, 4 mM ethylenediaminetetraacetic acid (EDTA), 5 mM benzamidine, 5 mM ACA, 1 mM ADP, and protease inhibitors (10 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A).</li>
		<li>Prepare Q-column buffer: 20 mM Tris-HCl pH 8.0, 4 mM EDTA, 10 mM MgSO4, 5 mM benzamidine, 5 mM ACA, and 1 mM ADP.</li>
		<li>Prepare Q-column elution buffer: Q-column buffer with 1 M NaCl.</li>
		<li>Prepare&#160;SEC&#160;buffer: 20 mM Tris-HCl pH 8.0, 10 mM MgSO4, 100 mM NaCl, 1 mM ADP.</li>
		<li>Prepare chloroform saturated with 2 M Tris-HCl pH 8.5. Mix chloroform with 2 M Tris-HCl pH 8.5 in approximately 1:1 ratio in a screw-cap bottle, shake, let the organic and aqueous phases separate, and measure pH in the upper aqueous layer with a strip of pH-indicator paper. Store at room temperature. Just before use, shake again and let the phases separate. Use the lower chloroform layer. 
		CAUTION: Chloroform is volatile and irritating to eyes and skin. Work in a fume hood. Use safety spectacles when shaking.</li>
	</ol>
	</li>
</ol>
2. Preparation of sub-mitochondrial Particles
<ol>
	<li>Resuspend mitochondrial vesicles (mitoplasts) isolated by hypotonic lysis25 from 1 x 1011 to 2 x 1011 cells of procyclic T. brucei in 5 mL of ice-cold buffer A. Keep the sample chilled until step 3.2.</li>
	<li>Determine the protein concentration in the suspension by the bicinchoninic acid (BCA) protein assay26 according to the manufacturer&#39;s instructions.
	<ol>
		<li>Use a bovine serum albumin (BSA) dilution series in ultrapure water to construct the standard curve. Dilute a small amount of sample 20 - 100 times with ultrapure water to fit into the range of BSA standards.</li>
		<li>Calculate the total protein amount in the sample and bring the protein concentration to 16 mg/mL by diluting it with additional buffer A.</li>
	</ol>
	</li>
	<li>Fragment mitoplasts into inverted vesicles and membrane pieces by sonication of the suspension 7x for 15 s with a total energy of 70 to 100 J per impulse with a microtip with a diameter of 3.9 mm. If the ultrasonic homogenizer does not display the energy output, start the optimization at 50% of the maximal power. Incubate the sample on ice for 30 s between impulses. After the sonication, the suspension becomes slightly darker.</li>
	<li>Sediment the membrane fragments by ultracentrifugation at 54,000 x g for 16 h or at 98,000 x g for 5 h at 4 &#176;C. Decant the supernatant and proceed with the chloroform extraction, or flash-freeze the sediment in liquid nitrogen and store it at -80 &#176;C.</li>
</ol>
3. Release of F1-ATPase from Membrane by Chloroform
<ol>
	<li>Resuspend the pellet of mitochondrial membranes in buffer B with the aid of a small Dounce homogenizer. Calculate the volume of buffer B based on the total amount of buffer A used in steps 2.1 and 2.2 using the following formula: volume (buffer B) = volume (buffer A) x 12/21. Transfer the suspension to a 15- or 50-mL conical tube.</li>
	<li>Remove the sample from ice and, from now on, keep the sample and all solutions to be used at room temperature.</li>
	<li>Add chloroform saturated with 2 M Tris-HCl pH 8.5; the volume of chloroform to be added equals half the volume of the suspension. Close the cap tightly. Shake it vigorously for exactly 20 s. Centrifuge it immediately at 8,400 x g for 5 min at room temperature.</li>
	<li>Transfer the upper cloudy aqueous phase to 1.6-mL microtubes. Add protease inhibitors (10 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A) to replace the inhibitors removed by the chloroform treatment. Centrifuge the samples at 13,000 x g for 30 min at room temperature. Transfer the supernatant to fresh microtubes and repeat the centrifugation to remove any insoluble material.</li>
</ol>
4. Anion-exchange Chromatography
<ol>
	<li>Equilibrate the 5-mL anion exchange (Q) column attached to a fast-protein liquid chromatography system with the Q-column buffer at a flow rate of 5 mL/min until the absorbance at 280 nm and the conductivity stabilize (approximately 50 mL of buffer).</li>
	<li>Load the supernatant from step 3.3 on the equilibrated column at a flow rate of 1 mL/min. Wait until the absorbance at 280 nm stabilizes at the background. Apply a 25-mL linear gradient of the Q-column elution buffer from 0% to 100% at a flow rate of 0.5 mL/min. Collect 1-mL fractions.</li>
	<li>Assay the individual fractions corresponding to the major elution peak for ATP hydrolytic activity by the Pullman ATPase assay2 at pH 8.0. Use 10 &#181;L of each fraction per 1 mL of reaction mixture.Pool the fractions that exhibit ATPase activity. Optionally, separate 10 &#181;L of each fraction on sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS-PAGE) and stain the gel by Coomassie Blue to visualize individual F1-ATPase subunits and contaminating proteins.</li>
	<li>Concentrate the pooled sample by membrane ultrafiltration using a spin column with a 100,000 MWCO PES filter to 200 - 500 &#181;L. Proceed to SEC or store the sample overnight at room temperature.</li>
</ol>
5. Size-exclusion Chromatography
<ol>
	<li>Equilibrate the SEC column attached to a liquid chromatography system with at least 48 mL (two column volumes) of the SEC buffer at a flow rate of 0.5 mL/min.</li>
	<li>Apply the sample on the column and run chromatography at a flow rate of 0.25 mL/min. Collect 0.25-mL fractions.</li>
	<li>Run 10 &#181;L of the fractions that correspond to the peaks of the UV280nm absorbance trace on SDS-PAGE and stain them by Coomassie Blue. The first major peak contains the F1-ATPase. Assay the fractions corresponding to this peak for the ATP hydrolytic activity and azide sensitivity by the Pullman ATPase assay. Determine the protein concentration by the BCA assay.</li>
	<li>Keep the purified F1-ATPase at room temperature and use it within 3 d after purification for downstream applications. Alternatively, concentrate the sample using a spin column with a 100,000 MWCO PES filter to &#62; 1.5 mg/mL, precipitate it by mixing it with saturated ammonium sulfate adjusted to pH 8.0 (1.2x the volume), and store it at 4 &#176;C.</li>
</ol>

<ol><li>Walker, J. E. Structure, mechanism and regulation of ATP synthases. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWalker%2C+JE+%22Mechanisms+of+Primary+Energy+Transduction+in+Biology%22">Mechanisms of Primary Energy Transduction in Biology</a>. Wikström, M. , The Royal Society of Chemistry. Croydon, UK. 338-373 (2017).</li>
<li>Pullman, M. E., Penefsky, H. S., Datta, A., Racker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+resolution+of+the+enzymes+catalyzing+oxidative+phosphorylation.+I.+Purification+and+properties+of+soluble+dinitrophenol-stimulated+adenosine+triphosphatase%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphatase.</a> The Journal of Biological Chemistry. 235, 3322-3329 (1960).</li>
<li>Schatz, G., Penefsky, H. S., Racker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+resolution+of+the+enzymes+catalyzing+oxidative+phosphorylation%5BTitle%5D)+AND+%22XIV.+The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Partial resolution of the enzymes catalyzing oxidative phosphorylation.</a> XIV. The Journal of Biological Chemistry. 242 (10), 2552-2560 (1967).</li>
<li>Racker, E., Horstman, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partial+resolution+of+the+enzymes+catalyzing+oxidative+phosphorylation.+13.+Structure+and+function+of+submitochondrial+particles+completely+resolved+with+respect+to+coupling+factor%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Partial resolution of the enzymes catalyzing oxidative phosphorylation. 13. Structure and function of submitochondrial particles completely resolved with respect to coupling factor.</a> The Journal of Biological Chemistry. 242 (10), 2547-2551 (1967).</li>
<li>Senior, A. E., Brooks, J. C. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/0003986170900305">Studies on the mitochondrial oligomycin-insensitive ATPase. I. An improved method of purification and the behavior of the enzyme in solutions of various depolymerizing agents.</a> Archives of Biochemistry and Biophysics. 140 (1), 257-266 (1970).</li>
<li>Tzagoloff, A., Meagher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+of+the+mitochondrial+membrane+system.+V.+Properties+of+a+dispersed+preparation+of+the+rutamycin-sensitive+adenosine+triphosphatase+of+yeast+mitochondria%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Assembly of the mitochondrial membrane system. V. Properties of a dispersed preparation of the rutamycin-sensitive adenosine triphosphatase of yeast mitochondria.</a> The Journal of Biological Chemistry. 246 (23), 7328-7336 (1971).</li>
<li>Beechey, R. B., Hubbard, S. A., Linnett, P. E., Mitchell, A. D., Munn, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+and+rapid+method+for+the+preparation+of+adenosine+triphosphatase+from+submitochondrial+particles%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles.</a> Biochemical Journal. 148 (3), 533-537 (1975).</li>
<li>Tyler, D. D., Webb, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+properties+of+the+adenosine+triphosphatase+released+from+the+liver+mitochondrial+membrane+by+chloroform%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform.</a> Biochemical Journal. 178 (2), 289-297 (1979).</li>
<li>Hack, E., Leaver, C. J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC555359/">The alpha-subunit of the maize F1-ATPase is synthesised in the mitochondrion.</a> The EMBO Journal. 2 (10), 1783-1789 (1983).</li>
<li>Dunn, P. P., Slabas, A. R., Moore, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+F1-ATPase+from+cuckoo-pint+%28Arum+maculatum%29+mitochondria.+A+comparison+of+subunit+composition+with+that+of+rat+liver+F1-ATPase%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Purification of F1-ATPase from cuckoo-pint (Arum maculatum) mitochondria. A comparison of subunit composition with that of rat liver F1-ATPase.</a> Biochemical Journal. 225 (3), 821-824 (1985).</li>
<li>Satre, M., Bof, M., Vignais, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+Escherichia+coli+adenosine+triphosphatase+with+aurovertin+and+citreoviridin%3A+inhibition+and+fluorescence+studies%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.</a> Journal of Bacteriology. 142 (3), 768-776 (1980).</li>
<li>Abrahams, J. P., Leslie, A. G., Lutter, R., Walker, J. E. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/8065448">Structure at 2.8 Å resolution of F1ATPase from bovine heart mitochondria.</a> Nature. 370 (6491), 621-628 (1994).</li>
<li>Lutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystallization+of+F1-ATPase+from+bovine+heart+mitochondria%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Crystallization of F1-ATPase from bovine heart mitochondria.</a> Journal of Molecular Biology. 229 (3), 787-790 (1993).</li>
<li>Kabaleeswaran, V., Puri, N., Walker, J. E., Leslie, A. G., Mueller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+features+of+the+rotary+catalytic+mechanism+revealed+in+the+structure+of+yeast+F1-ATPase%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1-ATPase.</a> The EMBO Journal. 25 (22), 5433-5442 (2006).</li>
<li>Shirakihara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+a+thermophilic+F1-ATPase+inhibited+by+an+epsilon-subunit%3A+deeper+insight+into+the+epsilon-inhibition+mechanism%5BTitle%5D)+AND+%22The+FEBS+Journal%22%5BJournal%5D)">Structure of a thermophilic F1-ATPase inhibited by an epsilon-subunit: deeper insight into the epsilon-inhibition mechanism.</a> The FEBS Journal. 282 (15), 2895-2913 (2015).</li>
<li>Stocker, A., Keis, S., Cook, G. M., Dimroth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification%2C+crystallization%2C+and+properties+of+F1-ATPase+complexes+from+the+thermoalkaliphilic+Bacillus+sp.+strain+TA2.A1%5BTitle%5D)+AND+%22Journal+of+Structural+Biology%22%5BJournal%5D)">Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.</a> Journal of Structural Biology. 152 (2), 140-145 (2005).</li>
<li>Ferguson, S. A., Cook, G. M., Montgomery, M. G., Leslie, A. G., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+thermoalkaliphilic+F1-ATPase+from+Caldalkalibacillus+thermarum%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (39), 10860-10865 (2016).</li>
<li>Cataldi de Flombaum, M. A., Frasch, A. C. C., Stoppani, A. O. M. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/0305049180901170">Adenosine triphosphatase from Trypanosoma cruzi: purification and properties.</a> Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 65 (1), 103-109 (1980).</li>
<li>Williams, N., Frank, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mitochondrial+ATP+synthase+of+Trypanosoma+brucei%3A+isolation+and+characterization+of+the+intact+F1+moiety%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">The mitochondrial ATP synthase of Trypanosoma brucei: isolation and characterization of the intact F1 moiety.</a> Molecular and Biochemical Parasitology. 43 (1), 125-132 (1990).</li>
<li>Higa, A. I., Cazzulo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mg2%2B-activated+adenosine+triphosphatase+from+Crithidia+fasciculata%3A+purification+and+inhibition+by+suramin+and+efrapeptin%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">Mg2+-activated adenosine triphosphatase from Crithidia fasciculata: purification and inhibition by suramin and efrapeptin.</a> Molecular and Biochemical Parasitology. 3 (6), 357-367 (1981).</li>
<li>Walker, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+structure+and+subunit+stoichiometry+of+F1-ATPase+from+bovine+mitochondria%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.</a> Journal of Molecular Biology. 184 (4), 677-701 (1985).</li>
<li>Mueller, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ni-chelate-affinity+purification+and+crystallization+of+the+yeast+mitochondrial+F1-ATPase%5BTitle%5D)+AND+%22Protein+Expression+and+Purification%22%5BJournal%5D)">Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.</a> Protein Expression and Purification. 37 (2), 479-485 (2004).</li>
<li>Gahura, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+F1-ATPase+from+Trypanosoma+brucei+is+elaborated+by+three+copies+of+an+additional+p18-subunit%5BTitle%5D)+AND+%22The+FEBS+Journal%22%5BJournal%5D)">The F1-ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.</a> The FEBS Journal. 285 (3), 614-628 (2018).</li>
<li>Montgomery, M. G., Gahura, O., Leslie, A. G. W., Zikova, A., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATP+synthase+from+Trypanosoma+brucei+has+an+elaborated+canonical+F1-domain+and+conventional+catalytic+sites%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2102-2107 (2018).</li>
<li>Schneider, A., Charriere, F., Pusnik, M., Horn, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+mitochondria+from+procyclic+Trypanosoma+brucei%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Isolation of mitochondria from procyclic Trypanosoma brucei.</a> Methods in Molecular Biology. 372, 67-80 (2007).</li>
<li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+protein+using+bicinchoninic+acid%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li>
<li>Wirtz, E., Leal, S., Ochatt, C., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tightly+regulated+inducible+expression+system+for+conditional+gene+knock-outs+and+dominant-negative+genetics+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.</a> Molecular and Biochemical Parasitology. 99 (1), 89-101 (1999).</li>
<li>Speijer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+respiratory+chain+from+cultured+Crithidia+fasciculata%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">Characterization of the respiratory chain from cultured Crithidia fasciculata.</a> Molecular and Biochemical Parasitology. 85 (2), 171-186 (1997).</li>
<li>Nelson, R. E., Aphasizheva, I., Falick, A. M., Nebohacova, M., Simpson, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15110463">The I-complex in Leishmania tarentolae is an uniquely-structured F1-ATPase.</a> Molecular and Biochemical Parasitology. 135 (2), 221-224 (2004).</li>
<li>Carbajo, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+the+N-terminal+domain+of+the+OSCP+subunit+of+bovine+F1Fo-ATP+synthase+interacts+with+the+N-terminal+region+of+an+alpha+subunit%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">How the N-terminal domain of the OSCP subunit of bovine F1Fo-ATP synthase interacts with the N-terminal region of an alpha subunit.</a> Journal of Molecular Biology. 368 (2), 310-318 (2007).</li>
<li>Bowler, M. W., Montgomery, M. G., Leslie, A. G., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ground+state+structure+of+F1-ATPase+from+bovine+heart+mitochondria+at+1.9+%C3%85+resolution%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 Å resolution.</a> The Journal of Biological Chemistry. 282 (19), 14238-14242 (2007).</li>
</ol>

The authors have nothing to disclose.

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 species13,14. 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...

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 or the reverse reaction occurs. F1-ATPase can be released experimentally from the membrane-intrinsic moiety while retaining its ability to hydrolyze, but not synthesize, ATP. The membrane-bound sector, called Fo, mediates protein translocation, which drives the rotation of the central part of the enzyme. The F1 and Fo sectors are connected by the&#160;central and peripheral stalks.
The first attempts to purify the F1-ATPase from budding yeast and bovine heart mitochondria date back to the 1960s. These protocols used extracted mitochondria, which were disrupted by sonication, fractionated by ammonium or protamine sulfate precipitation, followed by optional chromatography step(s) and heat treatment2,3,4,5,6. The purification was greatly improved and simplified by the use of chloroform, which readily releases the F1-ATPase from the mitochondrial membrane fragments7. The chloroform extraction was then used to extract F1-ATPases from various animal, plant, and bacterial sources (e.g., rat liver8, corn9, Arum maculatum10, and Escherichia coli11). Further purification of the chloroform-released F1-ATPase by affinity or size-exclusion chromatography (SEC) yielded a highly pure protein complex, which was suitable for high-resolution structure determination by X-ray crystallography, as documented by the structures of F1-ATPase from bovine heart12,13 and Saccharomyces cerevisiae14. F1-ATPase structures were also determined from organisms that are difficult to cultivate and, thus, the amount of the initial biological material was limited. In this case, the F1-ATPase subunits were artificially expressed and assembled into the complex in E. coli, and the whole heterologous enzyme was purified by affinity chromatography via a tagged subunit. Such approach led to the determination of F1-ATPase structures from two thermophilic bacterial species, Geobacillus stearothermophilus15 and Caldalkalibacillus thermarum16,17. However, this methodology is rather unsuitable for eukaryotic F1-ATPases since it relies on the prokaryotic protheosynthetic apparatus, posttranslational processing, and complex assembly.
The chloroform-based extraction was previously used to isolate F1-ATPases from unicellular digenetic parasites Trypanosoma cruzi18 and T. brucei19, important mammalian pathogens causing American and African trypanosomiases, respectively, and from monogenic insect parasite Crithidia fasciculata20. These purifications led only to a simple description of the F1-ATPases, since no downstream applications were used to fully characterize the composition, structure, and enzymatic properties of the complex. This article describes an optimized method for F1-ATPase purification from the cultured insect life cycle stage of T. brucei. The method is developed based on the established protocols for isolation of bovine and yeast F1-ATPases21,22. The procedure yields highly pure and homogeneous enzyme suitable for in vitro enzymatic and inhibitory assays, detailed proteomic characterization by mass spectrometry23, and structure determination24. The purification protocol and the knowledge of the F1-ATPase structure at the atomic level opens a possibility to design screens to identify small-molecule inhibitors, and aid in the development of new drugs against African trypanosomiases. Moreover, the protocol can be adapted to purify F1-ATPase from other organisms.

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

isolation of f1-atpase from the parasitic protist trypanosoma brucei

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

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&#39;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.

A typical purification (Figure 1) starts with mitochondrial vesicles (mitoplasts) isolated on the Percoll gradient from hypotonically lysed 1 x 1011 to 2 x 1011 procyclic T. brucei cells25 cultured in standard glucose-rich SDM-79 medium27. The mitoplasts are fragmented by sonication, spun, and the matrix-containing supernatant is discarded. Mitochondrial membranes are treated with ...

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.

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 ...

isolation-of-f1-atpase-from-the-parasitic-protist-trypanosoma-brucei

Research

JoVE Journal

Biochemistry

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of  F1FO ATPase complex and its modulation using the technique of patch clamp recording.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy.  As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput Gene Tagging in Trypanosoma brucei

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants.  This protocol describes a fast method for detecting and quantifying these populations.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

This study shows a detailed protocol to perform ultrastructure expansion microscopy in three in vitro life cycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. We include the optimized technique for cytoskeletal proteins and pan-proteome labeling.

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Learn to purify F1-ATPase from Trypanosoma brucei using classical biochemical methods. This protocol yields highly pure enzyme suitable for structural studies and drug discovery applications.

Isolation of F₁-ATPase from the Parasitic Protist Trypanosoma brucei

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