- Email: [email protected]
[44] Purification and properties of the ATPase inhibitor from bovine heart mitochondria
460
REVERSIBLEATP SYNTHASE (FoFl-ATPase)
[44]
reconstituted with Fj and OSCP except if the antibodies were preincubated with OSCP before the reconstitution. This means that the antibodies bind to OSCP integrated in the membrane without interfering with ATP synthesis or proton translocation. However, the OSCP-antibody complex cannot be used to reconstitute Fj with depleted SMP. Peptides Close to O S C P in the Complex
Cross-linking experiments made either by using the antibodies to identify the cross-linked products of OSCP or by direct photolabeling of the cross-linked products of OSCP revealed that major neighbors of OSCP were the a and/3 subunits of FI and at least two other peptides of about 30 and 24 kDa, not yet identified.
[44] Purification a n d P r o p e r t i e s of t h e A T P a s e Inhibitor from Bovine Heart Mitochondria By MAYNARD E. PULLMAN
Mitochondrial ATPase (F~) inhibitor is a small heat-stable protein that was first isolated from beef heart mitochondria.~ When complexed with either soluble or membrane-bound F1 ,L2 the protein inhibits the ATPase activity of the enzyme, but does not interfere with Frdependent oxidative phosphorylation. I Thus, the inhibitor-Fj complex, which exhibits no ATPase activity, retains an undiminished capacity to restore oxidative phosphorylation in Frdeficient submitochondrial particles (SMP).t The purification and properties of the inhibitor protein were described in an earlier volume of this series) Since then several other purification procedures for the beef heart, rat liver, yeast, and chloroplast inhibitor have been described. Most of these procedures have been patterned after the original purification procedure which exploited the heat and acid stability of the protein. References to these procedures together with an updated description of the properties of the inhibitor protein have been presented by Ernster et al. 4 in Volume 55 of this series. The procedure described here represents an improvement over the original procedure ~ M. 2 L. 3 G. 4 L.
E. Pullman and G. C. M o n r o y , J. Biol. Chem. 238, 3762 (1%3). L. H o r s t m a n a n d E. Racker, J. Biol. Chem. 2,45, 1316 (1970). C. M o n r o y a n d M. E. Pullman, this series, Vol. 10 [80]. Ernster, C. Carlsson, T. H. Undal, and K. N o r d e n b r a n d , this series, Vol. 55 [51].
METHODS IN ENZYMOLOGY, VOL. 126
Copyright © 1986by AcademicPress, Inc. All fights of reproduction in any form reserved.
[44]
ATPase INHIBITOR
461
with respect to yield and purity. Some newer aspects of the properties of the inhibitor are also described.
Assay Method
Principle The inhibitor content of fractions emerging from the purification procedure is determined by measuring the inhibition of the ATPase activity of either submitochondrial particles or soluble F1. The inhibitor does not inhibit ATPase in intact mitochondria. The assay system employing SMP has several advantages and is favored by most investigators. These advantages include the fact that SMP are easier to prepare than is soluble F I . 2 In addition the assay with SMP is less sensitive to s a l t s 1,2 and is reported to be 4 times more sensitive 2 than that described for the soluble enzyme. 3 The assay is carried out in two steps. The inhibitor is first incubated with submitochondrial particles, Mg z+, and ATP to permit the inhibitor to bind to the particle-bound ATPase. This is followed by an assay for the remaining ATPase activity. The procedure is similar to that described previously, z
Reagents 0.5 M Tris-SO4, pH 7.4 0.4 M sodium ATP, pH 7.4 0.4 M magnesium sulfate 0.05 M potassium phosphoenolpyruvate 5 10 mg/ml pyruvate kinase 6 0.2 M Bis-Tris-SO4, pH 6 . 5 7 0.025 M sodium ATP/0.025 M magnesium sulfate, pH 7.0 (ATP/Mg mixture) 0.25 M sucrose 1.0 M N a 2 S O 4 l0 mg/ml bovine serum albumin in 0.2 M Bis-Tris-SO4, pH 6.5 5 The monocyclohexylammonium salt of phosphoenolpyruvate was purchased from Sigma Chemical Company, St. Louis, MO and converted to the potassium salt as described in V. M. Clark and A. J. Kirby, Biochem. Prep. 11, 101 (1966). The potassium salt is also commercially available, but is about twice as expensive. 6 Pyruvate kinase is a crystalline suspension in ammonium sulfate (Boehringer-Mannheim) with a specific activity of 200. 7 [Bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane]; purchased from Sigma Chemical Company.
462
REVERSIBLEATP SYNTHASE(FoFrATPase)
[441
Mixed Medium A
A mixture sufficient for 25 assays contains 2.5 ml of Tris-SO4, 0.25 ml of ATP, 0.25 ml of MgSO4, 2.5 ml of phosphoenolpyruvate, and 0.75 ml water. This mixture is stable at - 2 0 ° for at least 6 months. Mixed Medium B
A mixture sufficient for 25 assays contains 6.25 ml of mixed medium A, 0.08 ml of pyruvate kinase, and 12.4 ml of water. Because of the uncertain stability of pyruvate kinase in the mixture, mixed medium B is prepared fresh and discarded at the end of the day. Inhibitor Diluent
To obtain reproducible measurements of inhibitor activity, particularly of highly purified preparations, it is essential to dilute the protein in a solution containing 20 mM Bris-Tris-SO4, pH 6.5, 0.2 M Na2SO4, and 1 mg/ml of bovine serum albumin. The diluent is prepared with 10 ml of Na2SO4, 5 ml of the bovine serum albumin solution, and 35 ml of water. Submitochondrial Particles
SMP are prepared by utilizing steps 1 and 2 and part of step 3 as described for the preparation of the soluble ATPase from beef heart mitochondria. 8 After incubation overnight in 0.1 M sucrose containing 4 mM ATP and 2 mM EDTA, pH 9.3, the particles are centrifuged at 30,000 rpm (106,000 g) for 90 min in the No. 30 rotor of a Spinco ultracentrifuge. The sedimented particles are resuspended in 0.1 M sucrose at a protein concentration of 35-40 mg/ml. The specific activity of the ATPase in these particles is between 2 and 4 and remains unchanged for at least 1 year when the preparation is stored at - 70°. To avoid unnecessary freezing and thawing, the particle suspension is stored in aliquots of 0.2 ml. Immediately before use, an aliquot is thawed and diluted with 0.25 M sucrose. Procedure
SMP containing 0.2-0.3 unit of ATPase activity (0.05-0.1 mg protein) are incubated for 5 min at 30° with 5-20 txl of the inhibitor sample in the presence of 5/xl of the ATP/Mg z+ mixture and 25/zl of Bis-Tris-SO4, pH 6.5. The volume is adjusted to 0.25 ml with 0.25 M sucrose. The final pH of the incubation mixture is between 6.3 and 6.5. The order of addition is s H. S. Penefsky, this series, Vol. 55 [33].
[44]
ATPase INHIBITOR
463
as follows: sucrose, buffer, inhibitor, SMP, and Mg2+-ATP. Because the inhibitor protein has a strong affinity for glass, the incubation is carried out in polystyrene test tubes. After 5 min of incubation at 30°, ATP hydrolysis is initiated by adding 0.75 ml of mixed medium B, prewarmed to 30°. After an additional 10 min of incubation, the reaction is terminated by the addition of 2 ml of 2.5% ammonium molybdate in 5 N H 2 S O 4 , and Pi is determined on the entire reaction mixture. 9 However, if the amount of protein in the inhibitor sample used exceeds 0.1 mg, the reaction is stopped with 0.1 ml 50% trichloroacetic acid. After centrifugation to remove the precipitated protein, Pi is measured on an aliquot of the supernatant. The samples are read at 660 m/z against a blank containing water in place of the assay mixture. The results are corrected for P~ in the reagents by carrying out an incubation without enzyme. The extent of ATP hydrolysis in the absence of added inhibitor is used to calculate the degree of inhibition in the inhibited samples. Titrations in which increasing amounts of inhibitor protein are added to the preincubation mixture should be carried out with each series of assays to ensure that the extent of inhibition is proportional to added inhibitor. The assay is not linear in the region above 50% inhibition. For reasons not entirely clear, a rectilinear titration curve is not always obtained even in the region below 50% inhibition. Thus, although acceptable for monitoring the progress of the purification procedure, the activity assay is less than satisfactory for more demanding quantitative measurements. A procedure using a radioimmunoassay has been developed for the inhibitor protein which is specific, sensitive, and more reliable for quantitative measurements. 10
Estimation of Protein The protein content of the mitochondrial suspension and of all fractions emerging from the purification procedure is measured spectrophotometricaUy 11 after diluting the samples in either concentrated formic acid (mitochondria) or buffer (soluble protein). The reference cell contains the corresponding solution, minus protein. The values obtained for the mitochondrial suspension agree very closely with those obtained by a biuret procedure modified for particulate protein.12 The biuret procedure was used to measure the protein content of submitochondrial particles. 9 K. Lohmann and L. Jendrassik, Biochem. Z. 178~ 419 (1926). ~0A. E. Otoadese, H. S. Penefsky, and M. E. Pullman, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 895 (1982). Complete details of the radioimmunoassay are described in a manuscript in preparation (1986). H O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). 12 E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. 223, 147 0956).
464
REVERSIBLEATP SYNTHASE(FoFi-ATPase)
[44]
Definition of Unit A unit of inhibitor activity is defined as the amount of protein which results in 50% inhibition of 0.2 unit of particulate ATPase under the specified assay conditions. One unit of ATPase activity is that amount of enzyme which hydrolyzes 1/zmol of ATP per minute under the specified assay conditions. Specific activity is expressed as units per milligram of protein. Purification of ATPase Inhibitor Beef heart mitochondria, 13 consisting of almost equal amounts of " h e a v y " and "light" layer mitochondria, is used as starting material. The mitochondrial preparation can be stored at - 6 0 ° in 0.25 sucrose/0.01 M T r i s - S O 4 , pH. 7.4 (sucrose/Tris) for at least 2 years without adversely affecting the outcome of the purification procedure. Twenty grams of mitochondria is worked up at a time and taken through the ammonium sulfate fractionation step. The ammonium sulfate fractions can be stockpiled at - 2 0 ° for at least 1 year without loss of inhibitor activity. Steps 1-4 of the purification procedure are conducted at 4 ° whereas steps 5-8 can be carried out at room temperature. All centrifugations are performed in a Sorval RC2-B centrifuge. Step 1: Washed Mitochondria. Prior to use, the mitochondrial suspension (60 to 70 mg/ml) is mixed with an equal volume of sucrose/Tris and centrifuged at 10,500 rpm (18,000 × g) in the GSA rotor for 20 min. The pellet is resuspended in sucrose/Tris to give the same final volume as the initial suspension and centrifuged as above. The latter procedure is repeated once more and the resulting mitochondrial pellet is resuspended in sucrose/Tris at a final protein concentration of 40 mg/ml. Step 2: Alkaline Extraction. To 500 ml (20 g protein) of the washed mitochondrial suspension is added an equal volume of cold glass-distilled water. While the suspension is stirred rapidly on a strong magnetic stirrer, the pH is rapidly adjusted to 11.5 with 25-30 ml of I N KOH. After 1 min the pH is readjusted to 7.5 with - 2 . 5 ml of 10 N acetic acid. The neutralized suspension is centrifuged for 10 min at 18,000 g, as above. Step 3: Trichloroacetic Acid Precipitation. To the somewhat turbid and yellow supernatant (-866 ml), 96 ml of 50% tricholoroacetic acid is added. After 10 min at 4 °, the precipitated protein is collected by centrifugation at 18,000 g for 10 min and suspended by homogenization in 150 ml distilled water. The water suspension of the precipitate (pH < 2) is adt3 p. V. Blair, this series, Vol. 10 [12].
[44]
ATPase INHIBITOR
465
justed to pH 11 with 6 N K O H and after 1 min is returned to pH 7.4 with 10 N acetic acid. After centrifugation at 18,000 g for 10 min, the slightly turbid supernatant solution is stored overnight at - 2 0 °. Step 4: Ammonium Sulfate Fractionation. After thawing, a large brown gelatinous precipitate, devoid of activity, is occasionally noticed and is removed by centrifugation. More often, only a slight turbidity appears and is ignored. To each 100 ml of solution, 29 g of ammonium sulfate is added, and after stirring on ice for 20 min the precipitate is removed by centrifugation and discarded. To each I00 ml of the supernatant solution is added 15.9 g of ammonium sulfate, and after 20 rain of stirring on ice, the precipitate is collected by centrifugation and dissolved in 50 mM Tris-SO4, pH 8.0, to give a final volume of 10-12 ml of a clear solution. The ammonium sulfate fraction may be stored at - 2 0 ° until all of the mitochondria have been brought to this stage. Step 5: Carboxymethyl (CM)-Cellulose Chromatography. Ammonium sulfate fractions obtained from 420 g of washed mitochondria are pooled and desalted on a column of Sephadex G-25 (fine), equilibrated with 10 raM" Tris-SO4, pH 8.5. The desalted preparation is applied at a flow rate of 2.5 ml/min to a CM-cellulose column (Whatman, CM-52; 1.6 × 6 cm), equilibrated with 10 mM Tris-SO4, pH 8.5. Under these conditions, almost all of the applied protein is recovered in the effluent. Although this step does not result in a significant increase in the specific activity of the preparation, it does remove all of the cytochrome c present in the fraction. Cytochrome c can be seen to form a tight red band at the top of the column. Omission of this step results in the contamination of the final inhibitor preparation by cytochrome c. Step 6: Diethylaminoethyl (DEAE)-Cellulose Chromatography. The effluent from the previous step is applied, without further treatment, to a DEAE-celluiose column (Whatman DE-52) (2.5 × 20 cm), equilibrated with 10 mM sodium pyrophosphate, pH 8.5. Column effluents and eluents are monitored at 280 nm with a UV detector attached to a recorder. The flow rate of the column is adjusted to 1.6 ml/min by means of a peristaltic pump. After the sample is applied, the column is washed with 10 mM pyrophosphate, pH 8.5, until the recorder returns to the baseline. The column is then eluted with 50 mM sodium pyrophosphate, pH 8.5. About 80% of the inhibitor activity applied is found in the next protein peak. Step 7: CM-Sephadex Chromatography. The step 6 inhibitor fraction (230 mg protein) is adjusted to pH 5.2 with 3 N acetic acid. The slightly turbid solution is centrifuged for 10 min at 12,000 g. The clear supernatant solution is applied at a pumping rate of 2.0 ml/min to a CM-25 Sephadex column (2.8 x 3.5 cm; bed volume, 22 ml) equilibrated with 10 mM sodium acetate, pH 5.2. After the sample is applied, the column is washed
466
REVERSIBLEATP SYNTHASE(FoFrATPase)
[44]
with 10 mM sodium acetate, pH 5.2, until the recorder returns to the baseline. The sample effluent plus the wash contains 80% of the applied protein but is devoid of inhibitor activity. The column is then eluted with 2.5 bed volumes of 10 mM sodium acetate, 100 mM sodium sulfate, pH 5.2. The eluate contains less than 1% of the protein and no inhibitor activity. Virtually all of the inhibitor activity applied is recovered when the column is subsequently eluted with 7 to 8 bed volumes of 10 mM sodium acetate, 1 M sodium sulfate, pH 5.2 (150 ml). Under these conditions the inhibitor protein is only slowly desorbed from the column. The rate of desorption is accelerated somewhat by periodically stopping the column flow for 30-60 min and then resuming collection. To concentrate and remove the inhibitor from the concentrated salt solution, the active fractions are pooled and the inhibitor protein is precipitated with 5% trichloroacetic acid at room temperature. The copius white precipitate is sedimented by centrifugation and dissolved in 10 mM PIPES, K ÷, pH 6.9. The pH of the protein solution is 2.2. With some preparations, the solution was slightly turbid at room temperature and somewhat more turbid at 4°. The insoluble protein, which exhibited no inhibitor activity, is removed by centrifugation at 4°. The crystal-clear supernatant solution is adjusted to pH 6.9 with KOH and applied to a Sephadex G-25 (fine) column, equilibrated with 10 mM PIPES, K ÷, pH 6.9, to ensure maximal removal of salt and trichloroacetic acid. A summary of the purification procedure is given in Table I. The values shown for the protein content of TABLE I SUMMARY OF PURIFICATION PROCEDUREa
Step I. W a s h e d mitochondria 2. Alkaline extract 3. Extract o f trichloroacetic acid precipitate 4. Desalted a m m o n i u m sulfate precipitate 5. CM-52 effluent 6. DE-52 eluent 7. C M - S e p h a d e x 8. Desalted trichloroacetic acid precipitate of step 7
Total protein (g) 420 23.1 7.7
Total units (x 106)
Specific activity (units/mg)
Yield (%)
-7.6 7.4
-329 961
-100 97
1.03
7.4
7,184
97
0.99 0.23 0.040 0.036
7.4 5.8 5.2 4.9
7,475 25,217 130,000 136,11 !
97 77 68 64
a T h e protein content of all fractions was m e a s u r e d spectrophotometrically, n This m e t h o d gives a low estimate for step 7 and step 8 fractions b e c a u s e of the unusually low content o f aromatic a m i n o acids in the inhibitor molecule (cf. text and Table If).
[44]
ATPase INHIBITOR
467
TABLE II PROTEIN CONCENTRATION OF THE STEP 8 FRACTION AS DETERMINED BY SEVERAL METHODS
Protein concentration Method
(mg/ml)
Dry weight ~5 Calculated from molar extinction coefficien04 Spectrophotometricl~ Lowry e t al. 16 Modified Lowry e t al. 17 Bicinchoninic acid ~8
5.52 5.46 0.81 6.60 6.76 4.40
all the fractions emerging from the purification procedure were determined spectrophotometrically, n This method gives a very low estimate of the purified protein (step 8) because of the unusually low content of aromatic amino acids in the molecule.14 To correct to the value obtained from the dry weight of the protein, 15 the value obtained from the spectrophotometric procedure II is multiplied by 6.81. Table I115-18 summarizes the protein concentration obtained for the step 8 fraction using several different methods. Protein concentration determined from the molar extinction coefficient of the inhibitor protein (1.68 × 103 M -~ cm-l)TM agrees closely with that based on its dry weight. Comments
Two Coomassie Brilliant Blue staining bands are observed when 100 /zg of protein 16 from the step 8 fraction are electrophoresed with sodium dodecyl sulfate (SDS) in polyacrylamide gels. 19The minor band, which is usually not visible when less than 50/zg of protein are applied to disk gels, represents about 3-5% of the total protein as determined from measurements of radioactivity in slices of gels containing 125I-labeled protein. Three lines of evidence suggest that the "impurity" is in fact a dimeric 14 B. Frangione, E. Rosenwasser, H. S. Penefsky, and M. E. Pullman, Proc. Natl. Acad. Sci. U . S , A . 78, 7403 (1981). ~5 M. E. Pullman and H. S. Penefsky, unpublished results. 16 O. H, Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 17 G. L. Peterson, Anal. Biochem. 83, 346 (1977). 18 Pierce Chemical Co., Previews, Oct. (1984). 29 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
468
REVERSIBLEATP SYNTHASE(FoFrATPase)
[44]
form of the inhibitor protein. It has a molecular weight of 18,500 (estimated from its migration in SDS-gel electrophoresis), 2° which is approximately twice that of the monomer form.14 Moreover, eluates of the gel band inhibit the ATPase activity of SMP and show strong cross-reactivity with a specific antibody raised against the inhibitor protein) ° Evidence for multiple molecular species of the beef heart, 21 rat liver, 22 and yeast 23 inhibitor proteins has been presented previously. In these cases, however, the multimeric forms appear to arise from the spontaneous aggregation of the protein, since interconversion of one form to another can be affected in vitro by salt zl and SDS. 22
Properties
Amino Acid Sequence The complete amino acid sequence of the beef heart inhibitor protein has been determined. 14The molecule contains 84 residues, accounting for a molecular weight of 9578. Lysine, arginine, glutamic acid, and aspartic acid comprise nearly 50% of the total amino acid residues, whereas apolar residues are present in low amounts. The polarity index calculated according to Capaldi and Vanderkooi 24 is 66%, making it considerably more hydrophilic than average proteins. The 40 charged amino acids are distributed along the chain in clusters which tend to occur with some regularity. A section of the chain, located at the COOH terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain contains all of the five histidines present in the molecule. The presence of a formyl-blocking group at the NH2 terminus has been found by some, 25 but not all investigators? 4,26 A comparison of the bovine heart inhibitor with the corresponding protein from the yeast Saccharomyces cerevisiae has revealed significant sequence homology between the two proteins. 25,27,28 On the other hand, 20 H. Reinheimer and M. E. Pullman, unpublished results. 2t G. Klein, M. Satre, G. Zaccai, and P. V. Vignais, Biochim. Biophys. Acta 681, 226 (1982). zz S. Chan, H. L. Tsai, C. Mohls, and D. McNeilly, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1080 (1985). 23 E. Ebner and L. L. Maier, J. Biol. Chem. 252, 671 (1977). 24 R. A. Capaldi and G. Vanderkooi, Proc. Natl. Acad. Sci. U.S.A. 69, 930 (1972). 25 A. C. Dianoux, A. Tsugita, and M. Przybylski, FEBS Lett. 174, 151 (1984). Dr. John Walker, personal communication. 27 H. Matsubara, K. Inoue, T. Hashimoto, Y. Yoshida, and K. Tagawa, J. Biochem. 94, 315 (1983). 28 We have used the computer program FASTP [D. L. Lipman and W. R. Pearson, Science 227, 1435 (1985)] to compare the amino acid sequence of the bovine ATPase inhibitor with
[44]
ATPase INHIBITOR
469
the chloroplast 29 and Escherichia colP° ATPase inhibitors, which are thought to be identical with the e subunits of the corresponding ATPases, do not appear to be related in sequence to the bovine or yeast inhibitors.
Relationship of Structure to Functional Activity Measurements of the inhibitory activity of peptides isolated after partial digestion of the beef heart inhibitor with proteolytic enzymes indicate that the first 16 amino acid residues from the NH2 terminus are not required for activity. 25 However, removal of 22 residues from the NHz terminal end or - 1 0 amino acids from the COOH terminal end destroys the inhibitory activity. 25,3~ Modification of two histidine residues in the inhibitor molecule by diethyl pyrocarbonate, under conditions where neither tyrosine fluorescence nor immunoreactivity is altered, results in the loss of binding to and inhibition of membrane-bound Fi.32
Functional Cross-Reactivity of ATPase Inhibitors A summary of the cross-reactivity of ATPase inhibitors from various sources has been presented in a previous volume of this series. 4 Since then, some additional information in this area has become available. For example, the bovine heart inhibitor fails to inhibit the purified E. coli ATPase. 33 Although the ATPase inhibitor purified from the yeast S. cerevisiae inhibits the ATPase activity of beef heart SMP, 34 it is 20 times less effective than the homologous beef heart inhibitor. 2° On the other hand, the beef heart and S. cerevisiae inhibitors appear to be almost equally effective in inhibiting the ATPase activity in yeast and rat liver each of the sequences in the National Biomedical Research Foundation protein sequence library. The best initial and optimized score was obtained with the ATPase inhibitor from S. cerevisiae.
29 E. T. Krebbers, I. M. Larrinua, L. Mclntosh, and L. Bogorad, Nucleic Acids Res. 10, 4985 (1982). 30 M. Saraste, N. J. Gay, A. Eberle, W. J. Runswick, and J. E. Walker, Nucleic Acids Res. 9, 5287 (1981). 3~ A. C. Dianoux, A. Tsugita, G. Klein, and P. V. Vignais, FEBS Lett. 140, 223 (1982). 3z D. A. Haake, A. E. Otoadese, H. S. Penefsky, and M. E. Pullman, unpublished observations. 33 A. E. Otoadese and M. E. Pullman, unpublished observations. The activity of purified E. coli ATPase, a gift from Dr. M. Futai, was unaffected by amounts of bovine ATPase inhibitor which were 20 times greater than that required to inhibit bovine F~ by 50%. 34 M. Tuena de Gomez-Puyou, U. Miller, J. Devars, A. Nava, and G. Dreyfus, FEBS Lett. 146, 168 (1982).
470
REVERSIBLEATP SYNTHASE(FoFvATPase)
[45]
SMP. 34 The ATPase inhibitor isolated from dog heart mitochondria is -30% as effective as the beef heart inhibitor on beef heart SMP2 ° Immunological Cross-Reactivity
Although comparisons of amino acid sequence indicate a genetic relationship between the mitochondrial ATPase inhibitors from S. cerevisiae and beef heart, their immunological characteristics differ. Antibodies against the bovine 2°,34and yeast 34inhibitor protein do not cross-react with the heterologous yeast and beef proteins, respectively. Antibodies to the yeast and bovine inhibitors also fail to react with the inhibitor from rat liver mitochondria) 4 However, dog heart inhibitor does cross-react with the antibody against the beef heart inhibitor. 2°
[45] P u r i f i c a t i o n o f t h e P r o t o n - T r a n s l o c a t i n g A T P a s e f r o m Rat Liver Mitochondria Using the Detergent 3 - [ ( 3 - C h o l a m i d o p r o p y l ) d i m e t h y l a m m o n i o ] - 1- p r o p a n e Sulfonate B y M A U R E E N W . M C E N E R Y a n d PETER L . PEDERSEN
The procedure that follows for the purification of the FoFI-ATPase from rat liver mitochondria is a modification of the original procedure by McEnery et al. 1 The important feature of this protocol which distinguishes it from earlier attempts at the purification of FoFrATPase complexes 2-7 is the use of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) developed by Hjelmeland 8 as an alternative to existing detergents. We have previously demonstrated that CHAPS appears to be the detergent of choice for the solubilization of the membrane-bound FoFrATPase complex, l In addition, by lowering the concentration of CHAPS present during velocity centrifugation in a sut M. W. McEnery, E. L. Buhle, U. Aebi, and P. L. Pedersen, J. Biol. Chem. 259, 4642 (1984). 2 j. W. Soper, G. L. Decker, and P. L. Pedersen, J. Biol. Chem. 254, 11170 (1979). 3 R. Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). D. L. Stiggall, Y. M. Galante, and Y. Hatefi, J. Biol. Chem. 252, 956 (1978). 5 R. Rott and N. Nelson, J. Biol. Chem. 256, 9224 (1981). 6 A. Tzagoloff and P. Meagher, J. Biol. Chem. 246, 7328 (1971). 7 D. L. Foster and R. H. Fillingame, J. Biol. Chem. 254, 8230 (1979). 8 L. M. Hjelmeland, Proc. Natl. Acad. Sci. U,S.A. 77, 6368 (1980).
METHODS IN ENZYMOLOGY,VOL. 126
Copyright© 1986by AcademicPress. Inc. All rightsof reproductionin any formreserved.
REVERSIBLEATP SYNTHASE (FoFl-ATPase)
[44]
reconstituted with Fj and OSCP except if the antibodies were preincubated with OSCP before the reconstitution. This means that the antibodies bind to OSCP integrated in the membrane without interfering with ATP synthesis or proton translocation. However, the OSCP-antibody complex cannot be used to reconstitute Fj with depleted SMP. Peptides Close to O S C P in the Complex
Cross-linking experiments made either by using the antibodies to identify the cross-linked products of OSCP or by direct photolabeling of the cross-linked products of OSCP revealed that major neighbors of OSCP were the a and/3 subunits of FI and at least two other peptides of about 30 and 24 kDa, not yet identified.
[44] Purification a n d P r o p e r t i e s of t h e A T P a s e Inhibitor from Bovine Heart Mitochondria By MAYNARD E. PULLMAN
Mitochondrial ATPase (F~) inhibitor is a small heat-stable protein that was first isolated from beef heart mitochondria.~ When complexed with either soluble or membrane-bound F1 ,L2 the protein inhibits the ATPase activity of the enzyme, but does not interfere with Frdependent oxidative phosphorylation. I Thus, the inhibitor-Fj complex, which exhibits no ATPase activity, retains an undiminished capacity to restore oxidative phosphorylation in Frdeficient submitochondrial particles (SMP).t The purification and properties of the inhibitor protein were described in an earlier volume of this series) Since then several other purification procedures for the beef heart, rat liver, yeast, and chloroplast inhibitor have been described. Most of these procedures have been patterned after the original purification procedure which exploited the heat and acid stability of the protein. References to these procedures together with an updated description of the properties of the inhibitor protein have been presented by Ernster et al. 4 in Volume 55 of this series. The procedure described here represents an improvement over the original procedure ~ M. 2 L. 3 G. 4 L.
E. Pullman and G. C. M o n r o y , J. Biol. Chem. 238, 3762 (1%3). L. H o r s t m a n a n d E. Racker, J. Biol. Chem. 2,45, 1316 (1970). C. M o n r o y a n d M. E. Pullman, this series, Vol. 10 [80]. Ernster, C. Carlsson, T. H. Undal, and K. N o r d e n b r a n d , this series, Vol. 55 [51].
METHODS IN ENZYMOLOGY, VOL. 126
Copyright © 1986by AcademicPress, Inc. All fights of reproduction in any form reserved.
[44]
ATPase INHIBITOR
461
with respect to yield and purity. Some newer aspects of the properties of the inhibitor are also described.
Assay Method
Principle The inhibitor content of fractions emerging from the purification procedure is determined by measuring the inhibition of the ATPase activity of either submitochondrial particles or soluble F1. The inhibitor does not inhibit ATPase in intact mitochondria. The assay system employing SMP has several advantages and is favored by most investigators. These advantages include the fact that SMP are easier to prepare than is soluble F I . 2 In addition the assay with SMP is less sensitive to s a l t s 1,2 and is reported to be 4 times more sensitive 2 than that described for the soluble enzyme. 3 The assay is carried out in two steps. The inhibitor is first incubated with submitochondrial particles, Mg z+, and ATP to permit the inhibitor to bind to the particle-bound ATPase. This is followed by an assay for the remaining ATPase activity. The procedure is similar to that described previously, z
Reagents 0.5 M Tris-SO4, pH 7.4 0.4 M sodium ATP, pH 7.4 0.4 M magnesium sulfate 0.05 M potassium phosphoenolpyruvate 5 10 mg/ml pyruvate kinase 6 0.2 M Bis-Tris-SO4, pH 6 . 5 7 0.025 M sodium ATP/0.025 M magnesium sulfate, pH 7.0 (ATP/Mg mixture) 0.25 M sucrose 1.0 M N a 2 S O 4 l0 mg/ml bovine serum albumin in 0.2 M Bis-Tris-SO4, pH 6.5 5 The monocyclohexylammonium salt of phosphoenolpyruvate was purchased from Sigma Chemical Company, St. Louis, MO and converted to the potassium salt as described in V. M. Clark and A. J. Kirby, Biochem. Prep. 11, 101 (1966). The potassium salt is also commercially available, but is about twice as expensive. 6 Pyruvate kinase is a crystalline suspension in ammonium sulfate (Boehringer-Mannheim) with a specific activity of 200. 7 [Bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane]; purchased from Sigma Chemical Company.
462
REVERSIBLEATP SYNTHASE(FoFrATPase)
[441
Mixed Medium A
A mixture sufficient for 25 assays contains 2.5 ml of Tris-SO4, 0.25 ml of ATP, 0.25 ml of MgSO4, 2.5 ml of phosphoenolpyruvate, and 0.75 ml water. This mixture is stable at - 2 0 ° for at least 6 months. Mixed Medium B
A mixture sufficient for 25 assays contains 6.25 ml of mixed medium A, 0.08 ml of pyruvate kinase, and 12.4 ml of water. Because of the uncertain stability of pyruvate kinase in the mixture, mixed medium B is prepared fresh and discarded at the end of the day. Inhibitor Diluent
To obtain reproducible measurements of inhibitor activity, particularly of highly purified preparations, it is essential to dilute the protein in a solution containing 20 mM Bris-Tris-SO4, pH 6.5, 0.2 M Na2SO4, and 1 mg/ml of bovine serum albumin. The diluent is prepared with 10 ml of Na2SO4, 5 ml of the bovine serum albumin solution, and 35 ml of water. Submitochondrial Particles
SMP are prepared by utilizing steps 1 and 2 and part of step 3 as described for the preparation of the soluble ATPase from beef heart mitochondria. 8 After incubation overnight in 0.1 M sucrose containing 4 mM ATP and 2 mM EDTA, pH 9.3, the particles are centrifuged at 30,000 rpm (106,000 g) for 90 min in the No. 30 rotor of a Spinco ultracentrifuge. The sedimented particles are resuspended in 0.1 M sucrose at a protein concentration of 35-40 mg/ml. The specific activity of the ATPase in these particles is between 2 and 4 and remains unchanged for at least 1 year when the preparation is stored at - 70°. To avoid unnecessary freezing and thawing, the particle suspension is stored in aliquots of 0.2 ml. Immediately before use, an aliquot is thawed and diluted with 0.25 M sucrose. Procedure
SMP containing 0.2-0.3 unit of ATPase activity (0.05-0.1 mg protein) are incubated for 5 min at 30° with 5-20 txl of the inhibitor sample in the presence of 5/xl of the ATP/Mg z+ mixture and 25/zl of Bis-Tris-SO4, pH 6.5. The volume is adjusted to 0.25 ml with 0.25 M sucrose. The final pH of the incubation mixture is between 6.3 and 6.5. The order of addition is s H. S. Penefsky, this series, Vol. 55 [33].
[44]
ATPase INHIBITOR
463
as follows: sucrose, buffer, inhibitor, SMP, and Mg2+-ATP. Because the inhibitor protein has a strong affinity for glass, the incubation is carried out in polystyrene test tubes. After 5 min of incubation at 30°, ATP hydrolysis is initiated by adding 0.75 ml of mixed medium B, prewarmed to 30°. After an additional 10 min of incubation, the reaction is terminated by the addition of 2 ml of 2.5% ammonium molybdate in 5 N H 2 S O 4 , and Pi is determined on the entire reaction mixture. 9 However, if the amount of protein in the inhibitor sample used exceeds 0.1 mg, the reaction is stopped with 0.1 ml 50% trichloroacetic acid. After centrifugation to remove the precipitated protein, Pi is measured on an aliquot of the supernatant. The samples are read at 660 m/z against a blank containing water in place of the assay mixture. The results are corrected for P~ in the reagents by carrying out an incubation without enzyme. The extent of ATP hydrolysis in the absence of added inhibitor is used to calculate the degree of inhibition in the inhibited samples. Titrations in which increasing amounts of inhibitor protein are added to the preincubation mixture should be carried out with each series of assays to ensure that the extent of inhibition is proportional to added inhibitor. The assay is not linear in the region above 50% inhibition. For reasons not entirely clear, a rectilinear titration curve is not always obtained even in the region below 50% inhibition. Thus, although acceptable for monitoring the progress of the purification procedure, the activity assay is less than satisfactory for more demanding quantitative measurements. A procedure using a radioimmunoassay has been developed for the inhibitor protein which is specific, sensitive, and more reliable for quantitative measurements. 10
Estimation of Protein The protein content of the mitochondrial suspension and of all fractions emerging from the purification procedure is measured spectrophotometricaUy 11 after diluting the samples in either concentrated formic acid (mitochondria) or buffer (soluble protein). The reference cell contains the corresponding solution, minus protein. The values obtained for the mitochondrial suspension agree very closely with those obtained by a biuret procedure modified for particulate protein.12 The biuret procedure was used to measure the protein content of submitochondrial particles. 9 K. Lohmann and L. Jendrassik, Biochem. Z. 178~ 419 (1926). ~0A. E. Otoadese, H. S. Penefsky, and M. E. Pullman, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 895 (1982). Complete details of the radioimmunoassay are described in a manuscript in preparation (1986). H O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). 12 E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. 223, 147 0956).
464
REVERSIBLEATP SYNTHASE(FoFi-ATPase)
[44]
Definition of Unit A unit of inhibitor activity is defined as the amount of protein which results in 50% inhibition of 0.2 unit of particulate ATPase under the specified assay conditions. One unit of ATPase activity is that amount of enzyme which hydrolyzes 1/zmol of ATP per minute under the specified assay conditions. Specific activity is expressed as units per milligram of protein. Purification of ATPase Inhibitor Beef heart mitochondria, 13 consisting of almost equal amounts of " h e a v y " and "light" layer mitochondria, is used as starting material. The mitochondrial preparation can be stored at - 6 0 ° in 0.25 sucrose/0.01 M T r i s - S O 4 , pH. 7.4 (sucrose/Tris) for at least 2 years without adversely affecting the outcome of the purification procedure. Twenty grams of mitochondria is worked up at a time and taken through the ammonium sulfate fractionation step. The ammonium sulfate fractions can be stockpiled at - 2 0 ° for at least 1 year without loss of inhibitor activity. Steps 1-4 of the purification procedure are conducted at 4 ° whereas steps 5-8 can be carried out at room temperature. All centrifugations are performed in a Sorval RC2-B centrifuge. Step 1: Washed Mitochondria. Prior to use, the mitochondrial suspension (60 to 70 mg/ml) is mixed with an equal volume of sucrose/Tris and centrifuged at 10,500 rpm (18,000 × g) in the GSA rotor for 20 min. The pellet is resuspended in sucrose/Tris to give the same final volume as the initial suspension and centrifuged as above. The latter procedure is repeated once more and the resulting mitochondrial pellet is resuspended in sucrose/Tris at a final protein concentration of 40 mg/ml. Step 2: Alkaline Extraction. To 500 ml (20 g protein) of the washed mitochondrial suspension is added an equal volume of cold glass-distilled water. While the suspension is stirred rapidly on a strong magnetic stirrer, the pH is rapidly adjusted to 11.5 with 25-30 ml of I N KOH. After 1 min the pH is readjusted to 7.5 with - 2 . 5 ml of 10 N acetic acid. The neutralized suspension is centrifuged for 10 min at 18,000 g, as above. Step 3: Trichloroacetic Acid Precipitation. To the somewhat turbid and yellow supernatant (-866 ml), 96 ml of 50% tricholoroacetic acid is added. After 10 min at 4 °, the precipitated protein is collected by centrifugation at 18,000 g for 10 min and suspended by homogenization in 150 ml distilled water. The water suspension of the precipitate (pH < 2) is adt3 p. V. Blair, this series, Vol. 10 [12].
[44]
ATPase INHIBITOR
465
justed to pH 11 with 6 N K O H and after 1 min is returned to pH 7.4 with 10 N acetic acid. After centrifugation at 18,000 g for 10 min, the slightly turbid supernatant solution is stored overnight at - 2 0 °. Step 4: Ammonium Sulfate Fractionation. After thawing, a large brown gelatinous precipitate, devoid of activity, is occasionally noticed and is removed by centrifugation. More often, only a slight turbidity appears and is ignored. To each 100 ml of solution, 29 g of ammonium sulfate is added, and after stirring on ice for 20 min the precipitate is removed by centrifugation and discarded. To each I00 ml of the supernatant solution is added 15.9 g of ammonium sulfate, and after 20 rain of stirring on ice, the precipitate is collected by centrifugation and dissolved in 50 mM Tris-SO4, pH 8.0, to give a final volume of 10-12 ml of a clear solution. The ammonium sulfate fraction may be stored at - 2 0 ° until all of the mitochondria have been brought to this stage. Step 5: Carboxymethyl (CM)-Cellulose Chromatography. Ammonium sulfate fractions obtained from 420 g of washed mitochondria are pooled and desalted on a column of Sephadex G-25 (fine), equilibrated with 10 raM" Tris-SO4, pH 8.5. The desalted preparation is applied at a flow rate of 2.5 ml/min to a CM-cellulose column (Whatman, CM-52; 1.6 × 6 cm), equilibrated with 10 mM Tris-SO4, pH 8.5. Under these conditions, almost all of the applied protein is recovered in the effluent. Although this step does not result in a significant increase in the specific activity of the preparation, it does remove all of the cytochrome c present in the fraction. Cytochrome c can be seen to form a tight red band at the top of the column. Omission of this step results in the contamination of the final inhibitor preparation by cytochrome c. Step 6: Diethylaminoethyl (DEAE)-Cellulose Chromatography. The effluent from the previous step is applied, without further treatment, to a DEAE-celluiose column (Whatman DE-52) (2.5 × 20 cm), equilibrated with 10 mM sodium pyrophosphate, pH 8.5. Column effluents and eluents are monitored at 280 nm with a UV detector attached to a recorder. The flow rate of the column is adjusted to 1.6 ml/min by means of a peristaltic pump. After the sample is applied, the column is washed with 10 mM pyrophosphate, pH 8.5, until the recorder returns to the baseline. The column is then eluted with 50 mM sodium pyrophosphate, pH 8.5. About 80% of the inhibitor activity applied is found in the next protein peak. Step 7: CM-Sephadex Chromatography. The step 6 inhibitor fraction (230 mg protein) is adjusted to pH 5.2 with 3 N acetic acid. The slightly turbid solution is centrifuged for 10 min at 12,000 g. The clear supernatant solution is applied at a pumping rate of 2.0 ml/min to a CM-25 Sephadex column (2.8 x 3.5 cm; bed volume, 22 ml) equilibrated with 10 mM sodium acetate, pH 5.2. After the sample is applied, the column is washed
466
REVERSIBLEATP SYNTHASE(FoFrATPase)
[44]
with 10 mM sodium acetate, pH 5.2, until the recorder returns to the baseline. The sample effluent plus the wash contains 80% of the applied protein but is devoid of inhibitor activity. The column is then eluted with 2.5 bed volumes of 10 mM sodium acetate, 100 mM sodium sulfate, pH 5.2. The eluate contains less than 1% of the protein and no inhibitor activity. Virtually all of the inhibitor activity applied is recovered when the column is subsequently eluted with 7 to 8 bed volumes of 10 mM sodium acetate, 1 M sodium sulfate, pH 5.2 (150 ml). Under these conditions the inhibitor protein is only slowly desorbed from the column. The rate of desorption is accelerated somewhat by periodically stopping the column flow for 30-60 min and then resuming collection. To concentrate and remove the inhibitor from the concentrated salt solution, the active fractions are pooled and the inhibitor protein is precipitated with 5% trichloroacetic acid at room temperature. The copius white precipitate is sedimented by centrifugation and dissolved in 10 mM PIPES, K ÷, pH 6.9. The pH of the protein solution is 2.2. With some preparations, the solution was slightly turbid at room temperature and somewhat more turbid at 4°. The insoluble protein, which exhibited no inhibitor activity, is removed by centrifugation at 4°. The crystal-clear supernatant solution is adjusted to pH 6.9 with KOH and applied to a Sephadex G-25 (fine) column, equilibrated with 10 mM PIPES, K ÷, pH 6.9, to ensure maximal removal of salt and trichloroacetic acid. A summary of the purification procedure is given in Table I. The values shown for the protein content of TABLE I SUMMARY OF PURIFICATION PROCEDUREa
Step I. W a s h e d mitochondria 2. Alkaline extract 3. Extract o f trichloroacetic acid precipitate 4. Desalted a m m o n i u m sulfate precipitate 5. CM-52 effluent 6. DE-52 eluent 7. C M - S e p h a d e x 8. Desalted trichloroacetic acid precipitate of step 7
Total protein (g) 420 23.1 7.7
Total units (x 106)
Specific activity (units/mg)
Yield (%)
-7.6 7.4
-329 961
-100 97
1.03
7.4
7,184
97
0.99 0.23 0.040 0.036
7.4 5.8 5.2 4.9
7,475 25,217 130,000 136,11 !
97 77 68 64
a T h e protein content of all fractions was m e a s u r e d spectrophotometrically, n This m e t h o d gives a low estimate for step 7 and step 8 fractions b e c a u s e of the unusually low content o f aromatic a m i n o acids in the inhibitor molecule (cf. text and Table If).
[44]
ATPase INHIBITOR
467
TABLE II PROTEIN CONCENTRATION OF THE STEP 8 FRACTION AS DETERMINED BY SEVERAL METHODS
Protein concentration Method
(mg/ml)
Dry weight ~5 Calculated from molar extinction coefficien04 Spectrophotometricl~ Lowry e t al. 16 Modified Lowry e t al. 17 Bicinchoninic acid ~8
5.52 5.46 0.81 6.60 6.76 4.40
all the fractions emerging from the purification procedure were determined spectrophotometrically, n This method gives a very low estimate of the purified protein (step 8) because of the unusually low content of aromatic amino acids in the molecule.14 To correct to the value obtained from the dry weight of the protein, 15 the value obtained from the spectrophotometric procedure II is multiplied by 6.81. Table I115-18 summarizes the protein concentration obtained for the step 8 fraction using several different methods. Protein concentration determined from the molar extinction coefficient of the inhibitor protein (1.68 × 103 M -~ cm-l)TM agrees closely with that based on its dry weight. Comments
Two Coomassie Brilliant Blue staining bands are observed when 100 /zg of protein 16 from the step 8 fraction are electrophoresed with sodium dodecyl sulfate (SDS) in polyacrylamide gels. 19The minor band, which is usually not visible when less than 50/zg of protein are applied to disk gels, represents about 3-5% of the total protein as determined from measurements of radioactivity in slices of gels containing 125I-labeled protein. Three lines of evidence suggest that the "impurity" is in fact a dimeric 14 B. Frangione, E. Rosenwasser, H. S. Penefsky, and M. E. Pullman, Proc. Natl. Acad. Sci. U . S , A . 78, 7403 (1981). ~5 M. E. Pullman and H. S. Penefsky, unpublished results. 16 O. H, Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 17 G. L. Peterson, Anal. Biochem. 83, 346 (1977). 18 Pierce Chemical Co., Previews, Oct. (1984). 29 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
468
REVERSIBLEATP SYNTHASE(FoFrATPase)
[44]
form of the inhibitor protein. It has a molecular weight of 18,500 (estimated from its migration in SDS-gel electrophoresis), 2° which is approximately twice that of the monomer form.14 Moreover, eluates of the gel band inhibit the ATPase activity of SMP and show strong cross-reactivity with a specific antibody raised against the inhibitor protein) ° Evidence for multiple molecular species of the beef heart, 21 rat liver, 22 and yeast 23 inhibitor proteins has been presented previously. In these cases, however, the multimeric forms appear to arise from the spontaneous aggregation of the protein, since interconversion of one form to another can be affected in vitro by salt zl and SDS. 22
Properties
Amino Acid Sequence The complete amino acid sequence of the beef heart inhibitor protein has been determined. 14The molecule contains 84 residues, accounting for a molecular weight of 9578. Lysine, arginine, glutamic acid, and aspartic acid comprise nearly 50% of the total amino acid residues, whereas apolar residues are present in low amounts. The polarity index calculated according to Capaldi and Vanderkooi 24 is 66%, making it considerably more hydrophilic than average proteins. The 40 charged amino acids are distributed along the chain in clusters which tend to occur with some regularity. A section of the chain, located at the COOH terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain contains all of the five histidines present in the molecule. The presence of a formyl-blocking group at the NH2 terminus has been found by some, 25 but not all investigators? 4,26 A comparison of the bovine heart inhibitor with the corresponding protein from the yeast Saccharomyces cerevisiae has revealed significant sequence homology between the two proteins. 25,27,28 On the other hand, 20 H. Reinheimer and M. E. Pullman, unpublished results. 2t G. Klein, M. Satre, G. Zaccai, and P. V. Vignais, Biochim. Biophys. Acta 681, 226 (1982). zz S. Chan, H. L. Tsai, C. Mohls, and D. McNeilly, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1080 (1985). 23 E. Ebner and L. L. Maier, J. Biol. Chem. 252, 671 (1977). 24 R. A. Capaldi and G. Vanderkooi, Proc. Natl. Acad. Sci. U.S.A. 69, 930 (1972). 25 A. C. Dianoux, A. Tsugita, and M. Przybylski, FEBS Lett. 174, 151 (1984). Dr. John Walker, personal communication. 27 H. Matsubara, K. Inoue, T. Hashimoto, Y. Yoshida, and K. Tagawa, J. Biochem. 94, 315 (1983). 28 We have used the computer program FASTP [D. L. Lipman and W. R. Pearson, Science 227, 1435 (1985)] to compare the amino acid sequence of the bovine ATPase inhibitor with
[44]
ATPase INHIBITOR
469
the chloroplast 29 and Escherichia colP° ATPase inhibitors, which are thought to be identical with the e subunits of the corresponding ATPases, do not appear to be related in sequence to the bovine or yeast inhibitors.
Relationship of Structure to Functional Activity Measurements of the inhibitory activity of peptides isolated after partial digestion of the beef heart inhibitor with proteolytic enzymes indicate that the first 16 amino acid residues from the NH2 terminus are not required for activity. 25 However, removal of 22 residues from the NHz terminal end or - 1 0 amino acids from the COOH terminal end destroys the inhibitory activity. 25,3~ Modification of two histidine residues in the inhibitor molecule by diethyl pyrocarbonate, under conditions where neither tyrosine fluorescence nor immunoreactivity is altered, results in the loss of binding to and inhibition of membrane-bound Fi.32
Functional Cross-Reactivity of ATPase Inhibitors A summary of the cross-reactivity of ATPase inhibitors from various sources has been presented in a previous volume of this series. 4 Since then, some additional information in this area has become available. For example, the bovine heart inhibitor fails to inhibit the purified E. coli ATPase. 33 Although the ATPase inhibitor purified from the yeast S. cerevisiae inhibits the ATPase activity of beef heart SMP, 34 it is 20 times less effective than the homologous beef heart inhibitor. 2° On the other hand, the beef heart and S. cerevisiae inhibitors appear to be almost equally effective in inhibiting the ATPase activity in yeast and rat liver each of the sequences in the National Biomedical Research Foundation protein sequence library. The best initial and optimized score was obtained with the ATPase inhibitor from S. cerevisiae.
29 E. T. Krebbers, I. M. Larrinua, L. Mclntosh, and L. Bogorad, Nucleic Acids Res. 10, 4985 (1982). 30 M. Saraste, N. J. Gay, A. Eberle, W. J. Runswick, and J. E. Walker, Nucleic Acids Res. 9, 5287 (1981). 3~ A. C. Dianoux, A. Tsugita, G. Klein, and P. V. Vignais, FEBS Lett. 140, 223 (1982). 3z D. A. Haake, A. E. Otoadese, H. S. Penefsky, and M. E. Pullman, unpublished observations. 33 A. E. Otoadese and M. E. Pullman, unpublished observations. The activity of purified E. coli ATPase, a gift from Dr. M. Futai, was unaffected by amounts of bovine ATPase inhibitor which were 20 times greater than that required to inhibit bovine F~ by 50%. 34 M. Tuena de Gomez-Puyou, U. Miller, J. Devars, A. Nava, and G. Dreyfus, FEBS Lett. 146, 168 (1982).
470
REVERSIBLEATP SYNTHASE(FoFvATPase)
[45]
SMP. 34 The ATPase inhibitor isolated from dog heart mitochondria is -30% as effective as the beef heart inhibitor on beef heart SMP2 ° Immunological Cross-Reactivity
Although comparisons of amino acid sequence indicate a genetic relationship between the mitochondrial ATPase inhibitors from S. cerevisiae and beef heart, their immunological characteristics differ. Antibodies against the bovine 2°,34and yeast 34inhibitor protein do not cross-react with the heterologous yeast and beef proteins, respectively. Antibodies to the yeast and bovine inhibitors also fail to react with the inhibitor from rat liver mitochondria) 4 However, dog heart inhibitor does cross-react with the antibody against the beef heart inhibitor. 2°
[45] P u r i f i c a t i o n o f t h e P r o t o n - T r a n s l o c a t i n g A T P a s e f r o m Rat Liver Mitochondria Using the Detergent 3 - [ ( 3 - C h o l a m i d o p r o p y l ) d i m e t h y l a m m o n i o ] - 1- p r o p a n e Sulfonate B y M A U R E E N W . M C E N E R Y a n d PETER L . PEDERSEN
The procedure that follows for the purification of the FoFI-ATPase from rat liver mitochondria is a modification of the original procedure by McEnery et al. 1 The important feature of this protocol which distinguishes it from earlier attempts at the purification of FoFrATPase complexes 2-7 is the use of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) developed by Hjelmeland 8 as an alternative to existing detergents. We have previously demonstrated that CHAPS appears to be the detergent of choice for the solubilization of the membrane-bound FoFrATPase complex, l In addition, by lowering the concentration of CHAPS present during velocity centrifugation in a sut M. W. McEnery, E. L. Buhle, U. Aebi, and P. L. Pedersen, J. Biol. Chem. 259, 4642 (1984). 2 j. W. Soper, G. L. Decker, and P. L. Pedersen, J. Biol. Chem. 254, 11170 (1979). 3 R. Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). D. L. Stiggall, Y. M. Galante, and Y. Hatefi, J. Biol. Chem. 252, 956 (1978). 5 R. Rott and N. Nelson, J. Biol. Chem. 256, 9224 (1981). 6 A. Tzagoloff and P. Meagher, J. Biol. Chem. 246, 7328 (1971). 7 D. L. Foster and R. H. Fillingame, J. Biol. Chem. 254, 8230 (1979). 8 L. M. Hjelmeland, Proc. Natl. Acad. Sci. U,S.A. 77, 6368 (1980).
METHODS IN ENZYMOLOGY,VOL. 126
Copyright© 1986by AcademicPress. Inc. All rightsof reproductionin any formreserved.