- Email: [email protected]
[35] Assay and purification of cytosolic factor required for cholera toxin activity
246
ASSAY OF TOXINS
[35]
[35] A s s a y a n d P u r i f i c a t i o n o f C y t o s o l i c F a c t o r R e q u i r e d for C h o l e r a T o x i n A c t i v i t y
By
MARILYN WOOLKALIS,
D.
MICHAEL GILL,
and
JENIFER COBURN
A cytosolic protein widespread in vertebrates is required in addition to a guanyl nucleotide triphosphate for the formation of a membrane complex that, in its turn, raises the ADP-ribosyltransferase activity of cholera toxin. The two steps can be separated in time. The cytosolic factor has been called CF ~ and its membrane-bound form S 2,3,4 or A R F ) Assaying the Cytosolic Factor
Pigeon Erythrocyte Membranes for CF Assay Pigeon erythrocyte membranes are particularly dependent on CF. They also have negligible NADase activity. Membranes can be prepared in bulk and stored until needed. Draw blood from the wing veins of pigeons into syringes containing heparin. Wash the cells several times with buffered saline [130 mM NaCI, 10 mM HEPES, pH 7.3, 0.01% NAN3, 0.01 trypsin inhibitor units (TIU)/ ml aprotinin]. Remove surface white cells by aspiration after each centrifugation and by passing the suspension once through a column of washed lambswool. Suspend the erythrocytes to a hematocrit of 50% and lyse them by immersing the tube in ethanol-dry ice. Thaw, dilute with 20 vol of buffered saline, and centrifuge. To digest the DNA in the crude ghost pellet, add an equal volume of 4 mM CaC12,6 U/ml micrococcal nuclease, and incubate at 37 ° for 15 min or until a small sample remains fluid when made 1% with SDS. Without washing, dispense the red slurry into 0.3-ml aliquots and store at - 7 0 °.
Standard CF Assay On the day of use, thaw a tube of nuclease-treated erythrocyte ghosts, dilute with 1.5 ml of saline, vortex well, and recover the membranes by centrifugation (12,000 g, 5 min). Wash again. Resuspend the pink membrane pellet in one pellet volume of buffered saline. Include 0.1% ovalbui K. 2 D. 3 D. 4 D. 5 R.
E n o m o t o and D. M. Gill, J. Biol. Chem. 255, 1252 (1980). M. Gill and R. Meren, J. Biol. Chem. 258, 11908 (1983). M. Gill and M. Woolkalis, this volume [34]. M. Gill and J. Coburn, Biochemistry 26, 6364 (1987). Kahn and A. G. Gilman, J. Biol. Chem. 261, 7906 (1986).
METHODS IN ENZYMOLOGY,VOL. 165
Copyright © 1988by Academic Press, Inc. All rightsof reproduction in any form reserved.
[35]
C Y T O S O L I C F A C T O R R E Q U I R E D BY C H O L E R A T O X I N
247
min in order to swamp differences in protein concentration between various samples of CF. On ice, add Gpp(NH)p or GTPyS to 200 ~ M and quickly dispense 10-/zl portions to cold tubes that already contain 10/zl, including the test CF and appropriate buffer to standardize the salt concentration and volume. Incubate 15 min at 37° and return to ice. To each tube add 10/~1 of a mixture of 20/zg/ml activated cholera toxin, 3 20 mM thymidine, 15 /xM [32p]NAD, and 15% (w/v) polyethylene glycol 8000. Incubate at 25 ° for 20 min, then dilute with 1 ml of buffered saline. Recover the membranes by centrifuging at 15,000 g for 10 min. Discard the supernatant, make the pellet into a slurry by vigorous vortexing, and then dissolve it in 40/zl of gel sample buffer. For maximum sensitivity fractionate the entire pellet solution on an SDS-polyacrylamide gel (7.5-15% acrylamide) and quantify the [32p]ADP-ribosyl Gs. 3 For quantitation, compare the test CF with a standard preparation over a range of concentrations. Since crude CF often demonstrates inhibition as well as activation, it is important to use several concentrations of the unknown and to compare rising phases of the response curves. As a negative control use a CF solution that has been heated to 80° for 15 min.
Rapid Minigel Assay For rapid assay of column fractions, use 10/~1 of each fraction per assay but fractionate only I0/xl of the sample buffer solution of membranes. Use minigels (10 × 8 cm, 12.5% acrylamide; IDEA Scientific, Corvallis, OR). Include positive and negative controls. Set the supply to 100 V until the sample clears the stacking gel, then 250 V for about 1 hr. Stain for 5 min, destain for 1 to 2 hr, dry the gel (30 min), and prepare an autoradiograph overnight.
More Rapid TCA Assay An even faster alternative is to count the trichloroacetic acid-insoluble material. At the end of the 25° incubation, wash the membranes in buffered saline, resuspend in 100 ~1 of 1% serum albumin, precipitate with 10% TCA, filter on Whatman GFA glass fiber circles, and count. Beware, however, of using the TCA assay for crude CF which contains poly(ADPribose) polymerase. Partial Purification of CF from Bovine Testis
Supernatants Testis is a good source. Cool the testes of freshly slaughtered bulls on ice and conduct all subsequent steps of the purification at 0-4 °. Discard
248
ASSAY OF TOXINS
[35]
the tunica albuginea. Mince testicular tissue (500 g) and homogenize in aliquots in a cooled Waring blender in 1 liter of buffer A (see below for recipe). Centrifuge at 30,000 g for 15 min. Aspirate the floating lipid. Centrifuge the combined supernatants at 100,000 g for 2 hr. Again aspirate any residual lipid off the surface. Collect the clarified supernatants, avoiding the cloudy layer above the pellet, and store at - 7 0 °.
Column Fractionation: General Use polypropylene tubes to collect the fractions from all of the columns. The purification can be improved by analyzing fractions by SDS-polyacrylamide minigels (15% acrylamide, Coomassie blue staining) and selecting and pooling those fractions that minimize protein complexity. CF has a molecular size of 20,000-21,000 Mr. In the later stages when the protein concentration becomes very low, CF may be stabilized by mixing in additional protein, such as insulin, that can be readily resolved from CF. The recoveries from step to step are difficult to quantitate because CF of different purities gives response curves of different shapes and different plateaus.
DEAE-Sephadex Apply 200 ml of the I00,000 g supernatant to a DEAE-Sephadex column (2.6 x 40 cm) equilibrated in buffer A. This column removes material that interferes with the resolution of later columns; batch procedures have not proved effective and DEAE-Sephacel cannot be substituted. Develop the column by pumping buffer A at 15 ml/hr, collecting 7ml fractions. Assay portions of each sample for CF activity, using a mini-gel assay. The TCA assay should not be used at this stage because the earliest peak of incorporation represents fractions rich in poly(ADPribose) polymerase. This peak should be discarded. Pool the fractions with CF activity (200-300 ml) that represent the majority of the UVabsorbing material. CF purified to this stage is adequate for routine work.
QAE-Sephadex For further purification, concentrate the DEAE-Sephadex pool to 25 ml using an Amicon TCF10 unit with a YM10 filter, dialyze extensively against buffer B, and centrifuge at 30,000 g. Apply to a QAE-Sephadex column (2.6 x 40 cm) equilibrated in buffer B. Pump at 30 ml/hr with buffer B, collecting 7-ml fractions. CF elutes at the trailing shoulder of the second major protein peak. Concentrate the active fractions to 5-10 ml as above.
[35]
CYTOSOLIC FACTOR REQUIRED BY CHOLERA TOXIN
249
Gel Filtration Apply the concentrate to a BioGel P-30 column (2.5 x 85 cm) equilibrated in buffer A. Elute at a constant 40 cm pressure head. Collect 3-ml fractions. Precalibrate the column with standard proteins: CF elutes between soybean trypsin inhibitor (20,000 Mr) and cytochrome ¢ (13,000 Mr). The TCA assay may be used: if there are two peaks the first peak represents poly(ADP-ribose) polymerase.
Hydroxylapatite Concentration is not usually necessary. Dialyze the P30 pool extensively against buffer C and apply to a BioGel HTP column (0.9 x 11 cm) equilibrated in the same buffer. Pump at 40 ml/hr and collect 3-ml fractions. Develop the column with 60 ml of buffer C, 60 ml of buffer D, and then a linear gradient formed from 95 ml each of buffers D and E. CF represents 10 to 50% of the protein eluted between 120 and 165 mM phosphate. Unfortunately, however, some of the main contaminants may have molecular sizes close to that of CF. Before assaying the fractions, neutralize and supplement with 100 mM NaC1. Pool the active fractions, concentrate 10-fold and change the medium to buffer A. Store aliquots at - 70°.
Buffers Employed in the CF Purification Buffer A: 50 mM HEPES, 10 mM sodium phosphate, 100 mM NaC1, 2 mM magnesium diacetate, 1 mM EGTA, 1 mM dithiothreitol, 0.01 TIU/ml aprotinin, 5/zg/ml leupeptin, pH 7.6 Buffer B: 50 mM AMPSO, 10 mM sodium phosphate, 50 mM NaCI, 2 mM magnesium diacetate, 1 mM EGTA, 1 mM dithiothreitol, 0.01 TIU/ml aprotinin, 5/xg/ml leupeptin, pH 9.0 Buffer C: 20 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Buffer D: 80 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Buffer E: 200 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Use degassed distilled water for preparing buffers C, D, and E. Acknowledgment This work was supported by NIH Grant AI 16928.
ASSAY OF TOXINS
[35]
[35] A s s a y a n d P u r i f i c a t i o n o f C y t o s o l i c F a c t o r R e q u i r e d for C h o l e r a T o x i n A c t i v i t y
By
MARILYN WOOLKALIS,
D.
MICHAEL GILL,
and
JENIFER COBURN
A cytosolic protein widespread in vertebrates is required in addition to a guanyl nucleotide triphosphate for the formation of a membrane complex that, in its turn, raises the ADP-ribosyltransferase activity of cholera toxin. The two steps can be separated in time. The cytosolic factor has been called CF ~ and its membrane-bound form S 2,3,4 or A R F ) Assaying the Cytosolic Factor
Pigeon Erythrocyte Membranes for CF Assay Pigeon erythrocyte membranes are particularly dependent on CF. They also have negligible NADase activity. Membranes can be prepared in bulk and stored until needed. Draw blood from the wing veins of pigeons into syringes containing heparin. Wash the cells several times with buffered saline [130 mM NaCI, 10 mM HEPES, pH 7.3, 0.01% NAN3, 0.01 trypsin inhibitor units (TIU)/ ml aprotinin]. Remove surface white cells by aspiration after each centrifugation and by passing the suspension once through a column of washed lambswool. Suspend the erythrocytes to a hematocrit of 50% and lyse them by immersing the tube in ethanol-dry ice. Thaw, dilute with 20 vol of buffered saline, and centrifuge. To digest the DNA in the crude ghost pellet, add an equal volume of 4 mM CaC12,6 U/ml micrococcal nuclease, and incubate at 37 ° for 15 min or until a small sample remains fluid when made 1% with SDS. Without washing, dispense the red slurry into 0.3-ml aliquots and store at - 7 0 °.
Standard CF Assay On the day of use, thaw a tube of nuclease-treated erythrocyte ghosts, dilute with 1.5 ml of saline, vortex well, and recover the membranes by centrifugation (12,000 g, 5 min). Wash again. Resuspend the pink membrane pellet in one pellet volume of buffered saline. Include 0.1% ovalbui K. 2 D. 3 D. 4 D. 5 R.
E n o m o t o and D. M. Gill, J. Biol. Chem. 255, 1252 (1980). M. Gill and R. Meren, J. Biol. Chem. 258, 11908 (1983). M. Gill and M. Woolkalis, this volume [34]. M. Gill and J. Coburn, Biochemistry 26, 6364 (1987). Kahn and A. G. Gilman, J. Biol. Chem. 261, 7906 (1986).
METHODS IN ENZYMOLOGY,VOL. 165
Copyright © 1988by Academic Press, Inc. All rightsof reproduction in any form reserved.
[35]
C Y T O S O L I C F A C T O R R E Q U I R E D BY C H O L E R A T O X I N
247
min in order to swamp differences in protein concentration between various samples of CF. On ice, add Gpp(NH)p or GTPyS to 200 ~ M and quickly dispense 10-/zl portions to cold tubes that already contain 10/zl, including the test CF and appropriate buffer to standardize the salt concentration and volume. Incubate 15 min at 37° and return to ice. To each tube add 10/~1 of a mixture of 20/zg/ml activated cholera toxin, 3 20 mM thymidine, 15 /xM [32p]NAD, and 15% (w/v) polyethylene glycol 8000. Incubate at 25 ° for 20 min, then dilute with 1 ml of buffered saline. Recover the membranes by centrifuging at 15,000 g for 10 min. Discard the supernatant, make the pellet into a slurry by vigorous vortexing, and then dissolve it in 40/zl of gel sample buffer. For maximum sensitivity fractionate the entire pellet solution on an SDS-polyacrylamide gel (7.5-15% acrylamide) and quantify the [32p]ADP-ribosyl Gs. 3 For quantitation, compare the test CF with a standard preparation over a range of concentrations. Since crude CF often demonstrates inhibition as well as activation, it is important to use several concentrations of the unknown and to compare rising phases of the response curves. As a negative control use a CF solution that has been heated to 80° for 15 min.
Rapid Minigel Assay For rapid assay of column fractions, use 10/~1 of each fraction per assay but fractionate only I0/xl of the sample buffer solution of membranes. Use minigels (10 × 8 cm, 12.5% acrylamide; IDEA Scientific, Corvallis, OR). Include positive and negative controls. Set the supply to 100 V until the sample clears the stacking gel, then 250 V for about 1 hr. Stain for 5 min, destain for 1 to 2 hr, dry the gel (30 min), and prepare an autoradiograph overnight.
More Rapid TCA Assay An even faster alternative is to count the trichloroacetic acid-insoluble material. At the end of the 25° incubation, wash the membranes in buffered saline, resuspend in 100 ~1 of 1% serum albumin, precipitate with 10% TCA, filter on Whatman GFA glass fiber circles, and count. Beware, however, of using the TCA assay for crude CF which contains poly(ADPribose) polymerase. Partial Purification of CF from Bovine Testis
Supernatants Testis is a good source. Cool the testes of freshly slaughtered bulls on ice and conduct all subsequent steps of the purification at 0-4 °. Discard
248
ASSAY OF TOXINS
[35]
the tunica albuginea. Mince testicular tissue (500 g) and homogenize in aliquots in a cooled Waring blender in 1 liter of buffer A (see below for recipe). Centrifuge at 30,000 g for 15 min. Aspirate the floating lipid. Centrifuge the combined supernatants at 100,000 g for 2 hr. Again aspirate any residual lipid off the surface. Collect the clarified supernatants, avoiding the cloudy layer above the pellet, and store at - 7 0 °.
Column Fractionation: General Use polypropylene tubes to collect the fractions from all of the columns. The purification can be improved by analyzing fractions by SDS-polyacrylamide minigels (15% acrylamide, Coomassie blue staining) and selecting and pooling those fractions that minimize protein complexity. CF has a molecular size of 20,000-21,000 Mr. In the later stages when the protein concentration becomes very low, CF may be stabilized by mixing in additional protein, such as insulin, that can be readily resolved from CF. The recoveries from step to step are difficult to quantitate because CF of different purities gives response curves of different shapes and different plateaus.
DEAE-Sephadex Apply 200 ml of the I00,000 g supernatant to a DEAE-Sephadex column (2.6 x 40 cm) equilibrated in buffer A. This column removes material that interferes with the resolution of later columns; batch procedures have not proved effective and DEAE-Sephacel cannot be substituted. Develop the column by pumping buffer A at 15 ml/hr, collecting 7ml fractions. Assay portions of each sample for CF activity, using a mini-gel assay. The TCA assay should not be used at this stage because the earliest peak of incorporation represents fractions rich in poly(ADPribose) polymerase. This peak should be discarded. Pool the fractions with CF activity (200-300 ml) that represent the majority of the UVabsorbing material. CF purified to this stage is adequate for routine work.
QAE-Sephadex For further purification, concentrate the DEAE-Sephadex pool to 25 ml using an Amicon TCF10 unit with a YM10 filter, dialyze extensively against buffer B, and centrifuge at 30,000 g. Apply to a QAE-Sephadex column (2.6 x 40 cm) equilibrated in buffer B. Pump at 30 ml/hr with buffer B, collecting 7-ml fractions. CF elutes at the trailing shoulder of the second major protein peak. Concentrate the active fractions to 5-10 ml as above.
[35]
CYTOSOLIC FACTOR REQUIRED BY CHOLERA TOXIN
249
Gel Filtration Apply the concentrate to a BioGel P-30 column (2.5 x 85 cm) equilibrated in buffer A. Elute at a constant 40 cm pressure head. Collect 3-ml fractions. Precalibrate the column with standard proteins: CF elutes between soybean trypsin inhibitor (20,000 Mr) and cytochrome ¢ (13,000 Mr). The TCA assay may be used: if there are two peaks the first peak represents poly(ADP-ribose) polymerase.
Hydroxylapatite Concentration is not usually necessary. Dialyze the P30 pool extensively against buffer C and apply to a BioGel HTP column (0.9 x 11 cm) equilibrated in the same buffer. Pump at 40 ml/hr and collect 3-ml fractions. Develop the column with 60 ml of buffer C, 60 ml of buffer D, and then a linear gradient formed from 95 ml each of buffers D and E. CF represents 10 to 50% of the protein eluted between 120 and 165 mM phosphate. Unfortunately, however, some of the main contaminants may have molecular sizes close to that of CF. Before assaying the fractions, neutralize and supplement with 100 mM NaC1. Pool the active fractions, concentrate 10-fold and change the medium to buffer A. Store aliquots at - 70°.
Buffers Employed in the CF Purification Buffer A: 50 mM HEPES, 10 mM sodium phosphate, 100 mM NaC1, 2 mM magnesium diacetate, 1 mM EGTA, 1 mM dithiothreitol, 0.01 TIU/ml aprotinin, 5/zg/ml leupeptin, pH 7.6 Buffer B: 50 mM AMPSO, 10 mM sodium phosphate, 50 mM NaCI, 2 mM magnesium diacetate, 1 mM EGTA, 1 mM dithiothreitol, 0.01 TIU/ml aprotinin, 5/xg/ml leupeptin, pH 9.0 Buffer C: 20 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Buffer D: 80 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Buffer E: 200 mM sodium phosphate, 1 mM dithiothreitol, pH 5.8 Use degassed distilled water for preparing buffers C, D, and E. Acknowledgment This work was supported by NIH Grant AI 16928.