- Email: [email protected]
[34] Purification of heme-containing soluble guanylyl cyclase
[34]
PURIFICATION
OF SOLUBLE
GUANYLYL
CYCLASE
377
Other Methods
After affinity chromatography and ion-exchange steps, Radany et al. 13 used a continuously eluting preparative polyacrylamide gel electrophoresis system as a final step in the purification of the enzyme (see [31], this volume). Recoveries obtained by this method are far superior to the concanavalin A-Sepharose elution described above, but the resulting enzyme is in the dephosphorylated form. Garbers12 used BioGel A 0.5m column (BioRad, Richmond, CA) chromatography as the final purification step, and Murad and co-workers 14used phenyl-agarose and wheat germ agglutinin A affinity columns after GTP-agarose and DEAE-Sephacel steps to copurify the guanylyl cyclase and atrial natriuretic factor receptor activity from rat lung (see also [37] and [38], this volume). A single band of 120,000 was obtained as judged by SDS-polyacrylamide gels. Starting with membranes obtained from rat adrenocortical carcinoma cells, Sharma and co-workers 15have purified guanylyl cyclase activity to apparent homogeneity by GTP-agarose and cGMP-agarose affinity columns. The resulting 180,000 protein possessed both guanylyl cyclase activity and atrial natriuretic factor receptor activity. T M
[34] P u r i f i c a t i o n of H e m e - C o n t a i n i n g Soluble Guanylyl Cyclase B y A L E X A N D E R MI3LSCH a n d RUPERT GERZER
Introduction
Guanylyl cyclases [EC 4.6.1.2, guanylate cyclase, GTP pyrophosphate-lyase (cyclizing)] catalyze the formation ofY,5'-cyclic GMP (cGMP) from 5'-GTP. In vertebrates, at least two different forms, particulate and cytosolic guanylyl cyclase, exist (for reviews, see Refs. 1 and 2). The cytosolic enzyme from bovine l u n g , 3'4 r a t lung,5 and rat liver6 and the I F. Murad, S. A. Waldman, R. R. Fiscus, and R. M. Rapoport, J. Cardiovasc. Pharmacol. 8 (Suppl. 8), $57 (1986). 2 j. Tremblay, R. Gerzer, and P. Hamet, Adv. Second Messenger Phosphoprotein Res. 22, 319 (1988). 3 R. Gerzer, F. Hofmann, and G. Schultz, Eur. J. Biochem. 116, 479 (1981). 4 L. J. Ignarro, K. S. Wood, and M. S. Wolin, Proc. Natl. Acad. Sci. U.S.A. 79, 2870 (1982). 5 j. A. Lewicki, H. J. Brandwein, C. K. Mittal, W. P. Arnold, and F. Murad, J. Cyclic Nucleotide Res. 8, 17 (1982). 6 p. A. Craven and F. R. DeRubertis, Biochim. Biophys. Acta 745, 310 (1983).
METHODS IN ENZYMOLOGY, VOL. 195
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
378
GUANYLYLCYCLASE
[34]
particulate enzyme from sea urchin spermatozoa, 7rat adrenocortical carcinoma, 8 and rat lung have been purified to apparent homogeneity. 9 The particulate form from sea urchin spermatozoa is a glycoprotein with partial homology in its primary sequence to protein kinases. ~° The particulate guanylyl cyclase from mammals is probably different from the sea urchin sperm enzyme and is activated by the atrial natriuretic factor and copurifies with the receptor of this factor. L2 The soluble form from bovine lung is a hemoprotein consisting of two subunits of about 75 and 70 kDa, H,~2 with the smaller subunit exhibiting partial homology with the sea urchin spermatozoan enzyme. ~3 Soluble and particulate forms differ with respect to enzyme kinetics and susceptibility to activation and inhibition by biological and chemical compounds.~'2 The soluble heme-containing guanylyl cyclase from bovine lung is directly activated by nitric oxide released from nitric oxide-containing vasodilators or by endothelium-derived nitric oxide (EDRF) released from endothelial cells.ll'14'15 In this chapter we describe a procedure for obtaining highly purified, heme-containing soluble guanylyl cyclase from bovine lung. The original procedure for purification of soluble guanylyl cyclase from bovine lung 3 was modified to meet the requirements for larger amounts of this enzyme for EPR studies,~Z'16 sequencing, 13and the detection of EDRF. 14The original procedure is outlined in principle and the modifications described in detail.
Determination of Guanylyl Cyclase Activity Guanylyl cyclase is localized in individual fractions after each purification step by measuring the enzymatic conversion of [a-32p]GTP to [32p]cGMP. Briefly, aliquots of selected fractions are incubated for 10 rain at 37° in the presence and absence of 100/zM sodium nitroprusside in a 7 E. W. Radany, R. Gerzer, and D. L. Garbers, J. Biol. Chem. 258, 8346 (1983). s A. K. Paul, R. B. Marala, R. K. Jaiswal, and R. K. Sharma, Science 235, 1224 (1987). 9 L. J. Chang, B. J. Chang, S. A. Waldn~an, and F. Murad, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1677 (1985). 10 S. Singh, D. G. Lowe, D. S. Thorpe, H. Rodriguez, W. J. Kuang, L. J. Dangott, M. Chinkers, D. V. Goeddel, and D. L. Garbers, Nature (London) 334, 708 (1988). 11 R. Gerzer, E. B6hme, F. Hofmann, and G. Schultz, FEBS Left. 132, 71 (1981). 12A. Mtilsch, Ph.D. Thesis, University of Heidelberg, Heidelberg, West Germany (1986). 13 D. Koesling, J. Herz, H. Gausepohl, F. Niroomand, K. D. Hinsch, A. Mtilsch, E. B6hme, G. Schultz, and R. Frank, FEBS Lett. 239, 29 0988). 14R. M. J. Palmer, A. G. Ferrige, and S. Moncada, Nature (London) 327, 524 (1987). 15A. Mtilsch, E. B6hme, and R. Busse, Eur. J. Pharmacol. 135, 247 (1987). 16A. Mfilsch and E. B6hme, Naunyn-Schmiedebergs Arch. Pharmacol. 325 (Suppl.), R31 (1984).
[34]
PURIFICATION OF SOLUBLE GUANYLYL CYCLASE
379
total volume of 100/zl containing 30 mM triethanolamine hydrochloride, pH 7.4, 3 mM magnesium chloride, 3 mM glutathione, 0.2 mM (0.2/xCi) [a-a2p]GTP, 0.1 mM cGMP, 5 mM creatine phosphate, 5 units creatine phosphokinase, 1 mM 3-isobutyl-l-methylxanthine, and 0.1 mM EGTA. The reaction is stopped by addition of 0.4 ml of 120 mM zinc acetate and 0.5 ml of 120 mM sodium carbonate. After centrifugation (10 min at 10,000 g), formed [32p]cGMP is isolated by chromatography of the supernatant fraction on acid alumina. 15Recovery of cGMP is about 50% as determined by the addition of [3H]cGMP to representative samples.
Purification Procedure General Outline. After preparation of a crude tissue extract, soluble guanylyl cyclase is purified by sequential application of anion-exchange chromatography on DEAE-Sepharose, ammonium sulfate precipitation, affinity chromatography on Blue Sepharose, and preparative polyacrylamide gel electrophoresis.3 In the modified procedure, granular DEAESepharose is replaced by DEAE-paper cartridges (Zetaprep-3200, Cuno, Mainz, FRG), the washing procedure for Blue Sepharose is reduced t o one step, and a modified electrophoresis device is used.17 Step 1: Preparation of Tissue Extract. All steps are performed in a cold room. Four to five fresh bovine lungs (10-12 kg) are obtained from a local slaughterhouse and placed on ice for transportation. Connective tissue and fat are carefully removed. The lungs are then minced and homogenized in a Waring blendor (45 sec at 15,000 rpm) with homogenizing buffer (I liter per 500 g mince) consisting of 10 mM potassium phosphate, pH 6.5, 0.2 mM benzamidine, and 6 mM EDTA. Following centrifugation (30 min at 14,000 g) the supernatants are filtered through glass wool and diluted 1 : 1 with homogenization buffer containing 100 mM 2-mercaptoethanol and 2 mM EDTA. Step 2: Chromatography on DEAE-CeUulose. Five Zetaprep-3200 DEAE cartridges connected in series are preprocessed (flow rate 50 liters/ hr) according to the following scheme: Regenerate with (1) 50 liters of 0.5 M acetic acid, pH 2-3; (2) 50 liters of 0.1 M sodium phosphate, pH 8; (3) repeat (1) and (2); (4) preequilibrate with 32 liters of 0.15 M potassium phosphate, pH 7; (5) equilibrate with 100 liters of 10 mM potassium phosphate, pH 6.5. The crude tissue extract (about 50 liters) is applied to the cartridges (20 liters/hr) by pressure with a roller pump. After 1 hr the first cartridge is disconnected from the perfusion because of high flow 17 A. M01sch and R. Gerzer, this volume [31].
380
GUANYLYLCYCLASE
[34]
~20 E
15 E x
..,,,.., ~o
~' ,.~
I [
I--,
'
=
I I _f ~i
x, \
',
J71L!J
7.5" .is Z
""....
..--~-~-, ............
.: I
........I ~
-20
....
. _'~-'-~.
X
__ .10
..........
II / //
0
.........
J
7" ...... \
''~. . . . . . . . . . . . . . . .
6.5l -S §
....""'"
.......~ " _
I0
_
J "
6
2"0 fractions 30
0
40
FIG. 1. DEAE-cellulose chromatography of bovine lung supernatant on Zetaprep-3200 fixed-bed cartridges. Five cartridges are perfused in series (6 liters/hr). Note the increase in pH during the development of the cartridges with the potassium chloride gradient. Pool I is used for the next purification step.
resistance. After loading the remainder of the extract onto cartridges 2 to 5, all cartridges are washed individually for at least 1 hr with modified homogenizing buffer containing 0.2 mM EDTA and 50 mM 2-mercaptoethanol with reversed flow (20 liters/hr). As soon as the wash is colorless, the cartridges are connected in series, and guanylyl cyclase is eluted (6 liters/hr) with a lienar gradient in potassium chloride (6 liters modified homogenizing buffer to 6 liters of the same buffer containing 0.5 M potassium chloride) and subsequently with l0 liters of 0.5 M potassium chloride. The eluate is fractionated (0.5 liter), and absorbance at 280 nm (protein content), pH, conductivity, and guanylyl cyclase activity are measured in individual fractions (Fig. 1). Fractions containing guanylyl cyclase activity are pooled as indicated in Fig. 1. Step 3: Ammonium Sulfate Precipitation. Pool I from the DEAE eluate is saturated with solid ammonium sulfate to 50% (0.294 g/ml) by agitation with an overhead stirrer. The pH should not be allowed to decrease below 6.5 and may require adjustment with ammonium hydroxide. After stirring for 30 min the precipitate is collected by centrifugation (30 min at 14,000 g) and is redissolved in 0.5 liter dialysis buffer (10 mM triethanolamine hydrochloride, pH 6.5, 0.2 mM benzamidine, 0.2 mM EDTA, 50 mM 2-mercaptoethanol). The solution is dialyzed for 2 hr against 40 liters of 0.2 mM benzamidine, 0.2 mM EDTA, 50 mM 2-mercaptoethanol, and afterward against 40 liters of dialysis buffer until the conductivity of the dialyzate is below 1 mS (about 7 hr). Step 4: Chromatography on Blue Sepharose. The dialyzate is checked for conductivity and pH (which must be 6.5 -+ 0.1) and is stirred for 30 min with 2.5 liters Blue Sepharose (Sepharose CL-6B containing 1/zmol/
[34]
381
P U R I F I C A T I O N OF S O L U B L E G U A N Y L Y L CYCLASE
• r- r-I
! !
I I
:
I
I I
1 ol
•
/ ;~00t-o ~1- .~1-
1
":/PJ
',NN
i
i 11_ 0<
N O
30
35
40 45 FRACTION
50
55
FIG. 2. Chromatography of ammonium sulfate dialyzate on Blue Sepharose. The column (10 x 30 cm) is developed (0.5 liter/hr) with 3 liters of buffer containing0.5 mM GTP/MnC12, pH 7.5. Note the dissociation in the rise of the GTP concentration and the pH in the eluate, which results in elution of contaminating proteins before the guanylyl cyclase is desorbed. The peak of guanylyl cyclase activity coincides with the elution of a chromophore absorbing at 430 nm. Pool H is used for the next purification step.
ml covalently coupled Cibacron Blue F3G-A) preequilibrated with dialysis buffer. The gel is washed with 40 liters dialysis buffer on a Biichner funnel (keep the first 2 liters for a check of binding) and packed into a column (10 × 60 cm) by gravity. Guanylyl cyclase is eluted in one step (0.5 liter/hr) with 3 liters dialysis buffer with the pH adjusted to 7.5 and supplemented with 0.5 m M G T P and 0.5 m M manganese chloride. The eluate is continuously monitored for absorbance at 280 nm (for GTP) and 430 nm (for hemoprotein). It is fractionated (30-ml fractions), and guanylyl cyclase activity, protein (Coomassie blue method), and pH are measured in individual fractions (Fig. 2). Guanylyl cyclase activity elutes as soon as the p H in the eluate rises. Fractions containing guanylyl cyclase activity are pooled as indicated in Fig. 2 and concentrated by centrifugation in ultrafree concentration units (30-kDa cutoffpolysulfone membranes, Millipore, Bedford, MA). Step 5: Preparative Polyacrylarnide Gel Electrophoresis. The concentrated pool (pool H) from the preceding step is diluted with electrophoresis elution buffer (154 m M glycine, 20 m M Tris, 50 m M 2-mercaptoethanol, 0.2 m M EDTA, 0.2 m M benzamidine hydrochloride, p H 8.6) containing 4% sucrose and is finally concentrated to 8 ml. Preparative electrophoresis is performed as described elsewhere in this volume. 17 The finally eluted
382
GUANYLYLCYCLASE
[34]
TABLE I PURIFICATION OF SOLUBLE GUANYLYL CYCLASE FROM BOVINE LUNGa Specific activity (nmol/mg/min)
Total activity (nmol/min)
Recovery (%)
Step
Protein (mg)
÷SNP
-SNP
+SNP
-SNP
+SNP
-SNP
Supernatant DEAE-cellulose AS dialyzate Blue Sepharose Electrophoresis
240,000 15,000 6530 330 10
0.03 16.00 44.00 310.00 2140.00
0.003 0.400 1.000 6.000 38.000
7200 240,000 287,300 102,300 21,400
720 6000 6530 1980 380
-100 120 43 9
-100 109 33 6
a Data are from a representative purification. Guanylyl cyclase activity was determined as described in the text. + SNP, Enzyme activity with 100/xM sodium nitroprusside; - S N P , basal activity; AS, ammonium sulfate. The calculation of recovery starts with the DEAE-cellulose step, since guanylyl cyclase activity is inhibited in the supernatant by hemoproteins and other factors.
enzyme is stored in 50% glycerol at - 70° under an atmosphere of nitrogen. No loss of activity has been found during storage up to 1 year. Comments on Purification The modified procedure shown in Table I yields about 10 mg soluble guanylyl cyclase from 5 bovine lungs within 3 days. The advantage of the modified method as compared to the original procedure is speed and capacity. This is achieved by the Zetaprep-3200 DEAE cartridges, which allow high flow rates, by Blue Sepharose containing less covalently coupled Cibacron Blue per volume of Sepharose, which permits a simplified washing and elution procedure, and the upscaling of the preparative electrophoresis device. The loading and washing of the Blue Sepharose critically depend on the pH, as a pH below 6.4 leads to diminished activation of guanylyl cyclase by nitric oxide due to loss of the heme, H whereas at a pH above 6.6 the binding capacity decreases. 12 Also, the preparative polyacrylamide gel should not be overloaded with protein, as this results in clogging of the protein onto the gel surface. Though the enzyme was purified to apparent homogeneity according to analytical polyacrylamide gel electrophoresis (Fig. 3), as well as isoelectric focusing (pl 5.9), the specific activity with sodium nitroprusside (about 2.1 units/mg) was lower than reported previously. 3 Since the heine binding to the enzyme is very labile, 11 and the stimulation of soluble guanylyl cyclase depends on the presence of heme, 3'4'6'7 the lower activation by sodium nitroprusside may be due to the lower heme content of the present preparation. ~6According
[34]
PURIFICATION
383
OF SOLUBLE GUANYLYL CYCLASE
B
._11 121 314 •
~ ~,, ~,~
•
l
!
l
/
s'-
•
|
~tart
68 I
45 29
21 18 6 MG
, 10
I, Ill 11
, In (MG)
!i
21 18 12.3 6.3 Front MG
FIG. 3. Determination of the subunit composition of purified soluble guanylyl cyclase from bovine lung. Apparent relative electrophoretic mobilities are plotted versus In[molecular weight (x 10-3)] of marker proteins. Note the higher relative mobility of soluble guanylyl cyclase subunits in the SDS-urea gel, which may be due to carbamylation of the protein by cyanate, which is in equilibrium with urea. (A) SDS-polyacrylamide gradient gel (12-18% acrylamide) prepared according to U. K. Laemmli, Nature (London) 227, 680 (1970). Lane 1, 1 #g soluble guanylyl cyclase from the preparative electrophoresis step; lane 2, marker proteins. Silver staining was performed according to W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197 (1981). (B) SDS-urea-polyacrylamide gel (6% acrylamide) prepared according to W. W. Minuth and K. Tiedemann, Histochemistry 68, 147 (1980). Lanes 1 and 2, marker proteins; lanes 3 and 4; l0 and 5/~g, respectively, of soluble guanylyl cyclase from the preparative electrophoresis step. The gel was stained with Coomassie blue. MG, Molecular weight. to analytical p o l y a c r y l a m i d e gel electrophoresis, sucrose density centrifugation, and size-exclusion c h r o m a t o g r a p h y (AcA 34, Pharmacia, Piscataway, N J; T S K 3000), the native e n z y m e has an apparent molecular weight of 145,000. U n d e r denaturing conditions it dissociates into two subunits of 75,000 and 70,000 (Fig. 3). Addendum In recent preparations we used 10 liters of D E A E - S e p h a r o s e Fast Flow (Pharmacia, Freiburg, F R G ) instead of the Zetaprep cartridges, since the latter lose binding capacity after prolonged use. The lung extract was cleared by passage through a 10-/~m filter cartridge (Cuno) and was loaded (10 liters/hr) on a column (15 × 30 cm) of D E A E - S e p h a r o s e Fast Flow, which was subsequently w a s h e d for 1 hr with 30 liters of l0 m M potassium p h o s p h a t e , p H 7. Guanylyl cyclase was then eluted with a salt gradient as described above.
PURIFICATION
OF SOLUBLE
GUANYLYL
CYCLASE
377
Other Methods
After affinity chromatography and ion-exchange steps, Radany et al. 13 used a continuously eluting preparative polyacrylamide gel electrophoresis system as a final step in the purification of the enzyme (see [31], this volume). Recoveries obtained by this method are far superior to the concanavalin A-Sepharose elution described above, but the resulting enzyme is in the dephosphorylated form. Garbers12 used BioGel A 0.5m column (BioRad, Richmond, CA) chromatography as the final purification step, and Murad and co-workers 14used phenyl-agarose and wheat germ agglutinin A affinity columns after GTP-agarose and DEAE-Sephacel steps to copurify the guanylyl cyclase and atrial natriuretic factor receptor activity from rat lung (see also [37] and [38], this volume). A single band of 120,000 was obtained as judged by SDS-polyacrylamide gels. Starting with membranes obtained from rat adrenocortical carcinoma cells, Sharma and co-workers 15have purified guanylyl cyclase activity to apparent homogeneity by GTP-agarose and cGMP-agarose affinity columns. The resulting 180,000 protein possessed both guanylyl cyclase activity and atrial natriuretic factor receptor activity. T M
[34] P u r i f i c a t i o n of H e m e - C o n t a i n i n g Soluble Guanylyl Cyclase B y A L E X A N D E R MI3LSCH a n d RUPERT GERZER
Introduction
Guanylyl cyclases [EC 4.6.1.2, guanylate cyclase, GTP pyrophosphate-lyase (cyclizing)] catalyze the formation ofY,5'-cyclic GMP (cGMP) from 5'-GTP. In vertebrates, at least two different forms, particulate and cytosolic guanylyl cyclase, exist (for reviews, see Refs. 1 and 2). The cytosolic enzyme from bovine l u n g , 3'4 r a t lung,5 and rat liver6 and the I F. Murad, S. A. Waldman, R. R. Fiscus, and R. M. Rapoport, J. Cardiovasc. Pharmacol. 8 (Suppl. 8), $57 (1986). 2 j. Tremblay, R. Gerzer, and P. Hamet, Adv. Second Messenger Phosphoprotein Res. 22, 319 (1988). 3 R. Gerzer, F. Hofmann, and G. Schultz, Eur. J. Biochem. 116, 479 (1981). 4 L. J. Ignarro, K. S. Wood, and M. S. Wolin, Proc. Natl. Acad. Sci. U.S.A. 79, 2870 (1982). 5 j. A. Lewicki, H. J. Brandwein, C. K. Mittal, W. P. Arnold, and F. Murad, J. Cyclic Nucleotide Res. 8, 17 (1982). 6 p. A. Craven and F. R. DeRubertis, Biochim. Biophys. Acta 745, 310 (1983).
METHODS IN ENZYMOLOGY, VOL. 195
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
378
GUANYLYLCYCLASE
[34]
particulate enzyme from sea urchin spermatozoa, 7rat adrenocortical carcinoma, 8 and rat lung have been purified to apparent homogeneity. 9 The particulate form from sea urchin spermatozoa is a glycoprotein with partial homology in its primary sequence to protein kinases. ~° The particulate guanylyl cyclase from mammals is probably different from the sea urchin sperm enzyme and is activated by the atrial natriuretic factor and copurifies with the receptor of this factor. L2 The soluble form from bovine lung is a hemoprotein consisting of two subunits of about 75 and 70 kDa, H,~2 with the smaller subunit exhibiting partial homology with the sea urchin spermatozoan enzyme. ~3 Soluble and particulate forms differ with respect to enzyme kinetics and susceptibility to activation and inhibition by biological and chemical compounds.~'2 The soluble heme-containing guanylyl cyclase from bovine lung is directly activated by nitric oxide released from nitric oxide-containing vasodilators or by endothelium-derived nitric oxide (EDRF) released from endothelial cells.ll'14'15 In this chapter we describe a procedure for obtaining highly purified, heme-containing soluble guanylyl cyclase from bovine lung. The original procedure for purification of soluble guanylyl cyclase from bovine lung 3 was modified to meet the requirements for larger amounts of this enzyme for EPR studies,~Z'16 sequencing, 13and the detection of EDRF. 14The original procedure is outlined in principle and the modifications described in detail.
Determination of Guanylyl Cyclase Activity Guanylyl cyclase is localized in individual fractions after each purification step by measuring the enzymatic conversion of [a-32p]GTP to [32p]cGMP. Briefly, aliquots of selected fractions are incubated for 10 rain at 37° in the presence and absence of 100/zM sodium nitroprusside in a 7 E. W. Radany, R. Gerzer, and D. L. Garbers, J. Biol. Chem. 258, 8346 (1983). s A. K. Paul, R. B. Marala, R. K. Jaiswal, and R. K. Sharma, Science 235, 1224 (1987). 9 L. J. Chang, B. J. Chang, S. A. Waldn~an, and F. Murad, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1677 (1985). 10 S. Singh, D. G. Lowe, D. S. Thorpe, H. Rodriguez, W. J. Kuang, L. J. Dangott, M. Chinkers, D. V. Goeddel, and D. L. Garbers, Nature (London) 334, 708 (1988). 11 R. Gerzer, E. B6hme, F. Hofmann, and G. Schultz, FEBS Left. 132, 71 (1981). 12A. Mtilsch, Ph.D. Thesis, University of Heidelberg, Heidelberg, West Germany (1986). 13 D. Koesling, J. Herz, H. Gausepohl, F. Niroomand, K. D. Hinsch, A. Mtilsch, E. B6hme, G. Schultz, and R. Frank, FEBS Lett. 239, 29 0988). 14R. M. J. Palmer, A. G. Ferrige, and S. Moncada, Nature (London) 327, 524 (1987). 15A. Mtilsch, E. B6hme, and R. Busse, Eur. J. Pharmacol. 135, 247 (1987). 16A. Mfilsch and E. B6hme, Naunyn-Schmiedebergs Arch. Pharmacol. 325 (Suppl.), R31 (1984).
[34]
PURIFICATION OF SOLUBLE GUANYLYL CYCLASE
379
total volume of 100/zl containing 30 mM triethanolamine hydrochloride, pH 7.4, 3 mM magnesium chloride, 3 mM glutathione, 0.2 mM (0.2/xCi) [a-a2p]GTP, 0.1 mM cGMP, 5 mM creatine phosphate, 5 units creatine phosphokinase, 1 mM 3-isobutyl-l-methylxanthine, and 0.1 mM EGTA. The reaction is stopped by addition of 0.4 ml of 120 mM zinc acetate and 0.5 ml of 120 mM sodium carbonate. After centrifugation (10 min at 10,000 g), formed [32p]cGMP is isolated by chromatography of the supernatant fraction on acid alumina. 15Recovery of cGMP is about 50% as determined by the addition of [3H]cGMP to representative samples.
Purification Procedure General Outline. After preparation of a crude tissue extract, soluble guanylyl cyclase is purified by sequential application of anion-exchange chromatography on DEAE-Sepharose, ammonium sulfate precipitation, affinity chromatography on Blue Sepharose, and preparative polyacrylamide gel electrophoresis.3 In the modified procedure, granular DEAESepharose is replaced by DEAE-paper cartridges (Zetaprep-3200, Cuno, Mainz, FRG), the washing procedure for Blue Sepharose is reduced t o one step, and a modified electrophoresis device is used.17 Step 1: Preparation of Tissue Extract. All steps are performed in a cold room. Four to five fresh bovine lungs (10-12 kg) are obtained from a local slaughterhouse and placed on ice for transportation. Connective tissue and fat are carefully removed. The lungs are then minced and homogenized in a Waring blendor (45 sec at 15,000 rpm) with homogenizing buffer (I liter per 500 g mince) consisting of 10 mM potassium phosphate, pH 6.5, 0.2 mM benzamidine, and 6 mM EDTA. Following centrifugation (30 min at 14,000 g) the supernatants are filtered through glass wool and diluted 1 : 1 with homogenization buffer containing 100 mM 2-mercaptoethanol and 2 mM EDTA. Step 2: Chromatography on DEAE-CeUulose. Five Zetaprep-3200 DEAE cartridges connected in series are preprocessed (flow rate 50 liters/ hr) according to the following scheme: Regenerate with (1) 50 liters of 0.5 M acetic acid, pH 2-3; (2) 50 liters of 0.1 M sodium phosphate, pH 8; (3) repeat (1) and (2); (4) preequilibrate with 32 liters of 0.15 M potassium phosphate, pH 7; (5) equilibrate with 100 liters of 10 mM potassium phosphate, pH 6.5. The crude tissue extract (about 50 liters) is applied to the cartridges (20 liters/hr) by pressure with a roller pump. After 1 hr the first cartridge is disconnected from the perfusion because of high flow 17 A. M01sch and R. Gerzer, this volume [31].
380
GUANYLYLCYCLASE
[34]
~20 E
15 E x
..,,,.., ~o
~' ,.~
I [
I--,
'
=
I I _f ~i
x, \
',
J71L!J
7.5" .is Z
""....
..--~-~-, ............
.: I
........I ~
-20
....
. _'~-'-~.
X
__ .10
..........
II / //
0
.........
J
7" ...... \
''~. . . . . . . . . . . . . . . .
6.5l -S §
....""'"
.......~ " _
I0
_
J "
6
2"0 fractions 30
0
40
FIG. 1. DEAE-cellulose chromatography of bovine lung supernatant on Zetaprep-3200 fixed-bed cartridges. Five cartridges are perfused in series (6 liters/hr). Note the increase in pH during the development of the cartridges with the potassium chloride gradient. Pool I is used for the next purification step.
resistance. After loading the remainder of the extract onto cartridges 2 to 5, all cartridges are washed individually for at least 1 hr with modified homogenizing buffer containing 0.2 mM EDTA and 50 mM 2-mercaptoethanol with reversed flow (20 liters/hr). As soon as the wash is colorless, the cartridges are connected in series, and guanylyl cyclase is eluted (6 liters/hr) with a lienar gradient in potassium chloride (6 liters modified homogenizing buffer to 6 liters of the same buffer containing 0.5 M potassium chloride) and subsequently with l0 liters of 0.5 M potassium chloride. The eluate is fractionated (0.5 liter), and absorbance at 280 nm (protein content), pH, conductivity, and guanylyl cyclase activity are measured in individual fractions (Fig. 1). Fractions containing guanylyl cyclase activity are pooled as indicated in Fig. 1. Step 3: Ammonium Sulfate Precipitation. Pool I from the DEAE eluate is saturated with solid ammonium sulfate to 50% (0.294 g/ml) by agitation with an overhead stirrer. The pH should not be allowed to decrease below 6.5 and may require adjustment with ammonium hydroxide. After stirring for 30 min the precipitate is collected by centrifugation (30 min at 14,000 g) and is redissolved in 0.5 liter dialysis buffer (10 mM triethanolamine hydrochloride, pH 6.5, 0.2 mM benzamidine, 0.2 mM EDTA, 50 mM 2-mercaptoethanol). The solution is dialyzed for 2 hr against 40 liters of 0.2 mM benzamidine, 0.2 mM EDTA, 50 mM 2-mercaptoethanol, and afterward against 40 liters of dialysis buffer until the conductivity of the dialyzate is below 1 mS (about 7 hr). Step 4: Chromatography on Blue Sepharose. The dialyzate is checked for conductivity and pH (which must be 6.5 -+ 0.1) and is stirred for 30 min with 2.5 liters Blue Sepharose (Sepharose CL-6B containing 1/zmol/
[34]
381
P U R I F I C A T I O N OF S O L U B L E G U A N Y L Y L CYCLASE
• r- r-I
! !
I I
:
I
I I
1 ol
•
/ ;~00t-o ~1- .~1-
1
":/PJ
',NN
i
i 11_ 0<
N O
30
35
40 45 FRACTION
50
55
FIG. 2. Chromatography of ammonium sulfate dialyzate on Blue Sepharose. The column (10 x 30 cm) is developed (0.5 liter/hr) with 3 liters of buffer containing0.5 mM GTP/MnC12, pH 7.5. Note the dissociation in the rise of the GTP concentration and the pH in the eluate, which results in elution of contaminating proteins before the guanylyl cyclase is desorbed. The peak of guanylyl cyclase activity coincides with the elution of a chromophore absorbing at 430 nm. Pool H is used for the next purification step.
ml covalently coupled Cibacron Blue F3G-A) preequilibrated with dialysis buffer. The gel is washed with 40 liters dialysis buffer on a Biichner funnel (keep the first 2 liters for a check of binding) and packed into a column (10 × 60 cm) by gravity. Guanylyl cyclase is eluted in one step (0.5 liter/hr) with 3 liters dialysis buffer with the pH adjusted to 7.5 and supplemented with 0.5 m M G T P and 0.5 m M manganese chloride. The eluate is continuously monitored for absorbance at 280 nm (for GTP) and 430 nm (for hemoprotein). It is fractionated (30-ml fractions), and guanylyl cyclase activity, protein (Coomassie blue method), and pH are measured in individual fractions (Fig. 2). Guanylyl cyclase activity elutes as soon as the p H in the eluate rises. Fractions containing guanylyl cyclase activity are pooled as indicated in Fig. 2 and concentrated by centrifugation in ultrafree concentration units (30-kDa cutoffpolysulfone membranes, Millipore, Bedford, MA). Step 5: Preparative Polyacrylarnide Gel Electrophoresis. The concentrated pool (pool H) from the preceding step is diluted with electrophoresis elution buffer (154 m M glycine, 20 m M Tris, 50 m M 2-mercaptoethanol, 0.2 m M EDTA, 0.2 m M benzamidine hydrochloride, p H 8.6) containing 4% sucrose and is finally concentrated to 8 ml. Preparative electrophoresis is performed as described elsewhere in this volume. 17 The finally eluted
382
GUANYLYLCYCLASE
[34]
TABLE I PURIFICATION OF SOLUBLE GUANYLYL CYCLASE FROM BOVINE LUNGa Specific activity (nmol/mg/min)
Total activity (nmol/min)
Recovery (%)
Step
Protein (mg)
÷SNP
-SNP
+SNP
-SNP
+SNP
-SNP
Supernatant DEAE-cellulose AS dialyzate Blue Sepharose Electrophoresis
240,000 15,000 6530 330 10
0.03 16.00 44.00 310.00 2140.00
0.003 0.400 1.000 6.000 38.000
7200 240,000 287,300 102,300 21,400
720 6000 6530 1980 380
-100 120 43 9
-100 109 33 6
a Data are from a representative purification. Guanylyl cyclase activity was determined as described in the text. + SNP, Enzyme activity with 100/xM sodium nitroprusside; - S N P , basal activity; AS, ammonium sulfate. The calculation of recovery starts with the DEAE-cellulose step, since guanylyl cyclase activity is inhibited in the supernatant by hemoproteins and other factors.
enzyme is stored in 50% glycerol at - 70° under an atmosphere of nitrogen. No loss of activity has been found during storage up to 1 year. Comments on Purification The modified procedure shown in Table I yields about 10 mg soluble guanylyl cyclase from 5 bovine lungs within 3 days. The advantage of the modified method as compared to the original procedure is speed and capacity. This is achieved by the Zetaprep-3200 DEAE cartridges, which allow high flow rates, by Blue Sepharose containing less covalently coupled Cibacron Blue per volume of Sepharose, which permits a simplified washing and elution procedure, and the upscaling of the preparative electrophoresis device. The loading and washing of the Blue Sepharose critically depend on the pH, as a pH below 6.4 leads to diminished activation of guanylyl cyclase by nitric oxide due to loss of the heme, H whereas at a pH above 6.6 the binding capacity decreases. 12 Also, the preparative polyacrylamide gel should not be overloaded with protein, as this results in clogging of the protein onto the gel surface. Though the enzyme was purified to apparent homogeneity according to analytical polyacrylamide gel electrophoresis (Fig. 3), as well as isoelectric focusing (pl 5.9), the specific activity with sodium nitroprusside (about 2.1 units/mg) was lower than reported previously. 3 Since the heine binding to the enzyme is very labile, 11 and the stimulation of soluble guanylyl cyclase depends on the presence of heme, 3'4'6'7 the lower activation by sodium nitroprusside may be due to the lower heme content of the present preparation. ~6According
[34]
PURIFICATION
383
OF SOLUBLE GUANYLYL CYCLASE
B
._11 121 314 •
~ ~,, ~,~
•
l
!
l
/
s'-
•
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~tart
68 I
45 29
21 18 6 MG
, 10
I, Ill 11
, In (MG)
!i
21 18 12.3 6.3 Front MG
FIG. 3. Determination of the subunit composition of purified soluble guanylyl cyclase from bovine lung. Apparent relative electrophoretic mobilities are plotted versus In[molecular weight (x 10-3)] of marker proteins. Note the higher relative mobility of soluble guanylyl cyclase subunits in the SDS-urea gel, which may be due to carbamylation of the protein by cyanate, which is in equilibrium with urea. (A) SDS-polyacrylamide gradient gel (12-18% acrylamide) prepared according to U. K. Laemmli, Nature (London) 227, 680 (1970). Lane 1, 1 #g soluble guanylyl cyclase from the preparative electrophoresis step; lane 2, marker proteins. Silver staining was performed according to W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197 (1981). (B) SDS-urea-polyacrylamide gel (6% acrylamide) prepared according to W. W. Minuth and K. Tiedemann, Histochemistry 68, 147 (1980). Lanes 1 and 2, marker proteins; lanes 3 and 4; l0 and 5/~g, respectively, of soluble guanylyl cyclase from the preparative electrophoresis step. The gel was stained with Coomassie blue. MG, Molecular weight. to analytical p o l y a c r y l a m i d e gel electrophoresis, sucrose density centrifugation, and size-exclusion c h r o m a t o g r a p h y (AcA 34, Pharmacia, Piscataway, N J; T S K 3000), the native e n z y m e has an apparent molecular weight of 145,000. U n d e r denaturing conditions it dissociates into two subunits of 75,000 and 70,000 (Fig. 3). Addendum In recent preparations we used 10 liters of D E A E - S e p h a r o s e Fast Flow (Pharmacia, Freiburg, F R G ) instead of the Zetaprep cartridges, since the latter lose binding capacity after prolonged use. The lung extract was cleared by passage through a 10-/~m filter cartridge (Cuno) and was loaded (10 liters/hr) on a column (15 × 30 cm) of D E A E - S e p h a r o s e Fast Flow, which was subsequently w a s h e d for 1 hr with 30 liters of l0 m M potassium p h o s p h a t e , p H 7. Guanylyl cyclase was then eluted with a salt gradient as described above.