- Email: [email protected]
[13] Purification of PGH-PGE isomerase from sheep vesicular glands
84
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
[13]
reaction. The optimum glutathione concentration lies between 10-4 and 10-3 M. Without glutathione, no enzymic PGD2 formation could be demonstrated. pH Optimum. Incubations were done between pH 4 and 9.4 in the presence of 1 mM glutathione. Optimum conversion was obtained between pH 7 and 8. 3 Inhibitory Substances. A number of compounds were tested that might inhibit the isomerization either by interference with the substrate binding site or with the glutathione binding site of the enzyme. Only those compounds that can react with free SH groups, such as 15-hydroperoxyarachidonic acid and p-hydroxymercuribenzoate, were found to inhibit the conversion. 3
[13] Purification
of PGH-PGE Isomerase Vesicular Glands
from Sheep
By P. MOONEN, M. BUYTENHEK, and D. H. NUGTEREN For studies on prostaglandin biosynthesis, the microsomal fraction of sheep vesicular glands is more or less the "classical" starting material. The biosynthesis of prostaglandin E (PGE) 1 from certain unsaturated fatty acids is a membrane-bound process that requires two enzymes acting sequentially. 2,a The first enzyme, which catalyzes the conversion of certain essential fatty acids (e.g., arachidonic acid) into prostaglandin H (PGH) is referred to as PGH synthase. This protein has been fully purified both from bovine and sheep vesicular gland microsomes. 4 The second enzyme needs glutathione (GSH) for its activity and isomerizes the endoperoxy group of PGH to a 9-keto-, 11-hydroxyl arrangement (PGE). This socalled prostaglandin endoperoxide-E isomerase ( P G H - P G E isomerase, EC 5.3.99.3) has been partially purified from bovine vesicular gland microsomes. 5 In this contribution our approaches to purification of P G H PGE isomerase from sheep vesicular gland microsomes are described.
i For nomenclature of prostaglandins see N. A. Nelson, J. Med. Chem. 17, 911 (1974). 2 D. H. Nugteren and E. Hazelhof, Biochim. Biophys. Acta 326, 448 (1973). 3 T. Miyamoto, S. Yamamoto, and O. Hayaishi, Proc. Natl. Acad. Sci. U.S.A. 71, 3645 (1974). 4 F. J. van der Ouderaraa and M. Buytenhek, this volume [9]. 5 N. Ogino, T. Miyamoto, S. Yamamoto, and O. Hayaishi, J. Biol. Chem. 252, 890 (1977).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
85
Assay Method Principle. The endoperoxide used as substrate in the assay is not stable in aqueous medium: it has a half-life of about 10 min at pH 7.4 and 20°. The main degradation products are PGE and PGD in a ratio of about 3 : 1. Therefore, incubations are carried out for only 1 min with different amounts of enzyme and with a fixed amount of 14C-labeled endoperoxide. After quick acidification and extraction with ether, the products formed and the remaining endoperoxide are rapidly separated by silica gelHPTLC. Quantification of the enzyme activity is possible by scraping off the radioactive bands, counting the radioactivity, and calculating the nanomoles of PGE formed. Preparation of Substrate (see also this volume [12]). Purified prostaglandin endoperoxide synthase4 (4.5 mg of protein, specific activity 16.9 U/mg) was preincubated with 1 mg of hemin in 16 ml of 0.1 M Tris-HCl, pH 8, for 5 min at 0°. [1-14C]Arachidonic acid (Radiochemical Centre, Amersham, 23 × 106 dpm) was added to unlabeled arachidonic acid to a total of 9.0 mg. The fatty acid plus 3 mg of hydroquinone were dissolved in a 250-ml Erlenmeyer flask in 40 ml of 0.1 M Tris-0.1 M phosphate, 1 mM EDTA, pH 8.0. The turbid suspension was carefully saturated with air and placed in a water bath at 29°. The enzyme solution was warmed up and subsequently transferred to the fatty acid suspension; the mixture was then incubated for 2.5 min at 29°. The Erlenmeyer flask was shaken manually, and a stream of air was introduced into the flask. The reaction was terminated by pouring the mixture rapidly into 18 ml of 0.2 M citric acid plus 100 ml of ethyl acetate cooled in ethanol-solid CO2, immediately followed by thoroughly shaking. The reaction vessel was cooled so that the water phase just did not freeze. After separation of the two phases, the ethyl acetate phase was immediately decanted and the water phase was rapidly extracted with 25 ml of cooled ethyl acetate. The combined ethyl acetate phases were cooled to - 80° for 4 hr to freeze out the water, and then filtered at - 80° over a cooled glass filter G3 (15-40/zm). The precipitate was washed with 25 ml of cooled ethyl acetate. The filtered ethyl acetate was evaporated under reduced pressure to approximately 1 ml and spotted in a cold room as a 10-cm band on a 20 × 20 cm 0.5 mm-thick silica gel 60 F254 plate (Merck); 10/zg of prostaglandin B~ or B2 had been spotted beforehand as a reference. The plate was developed without delay at 4° for 1 hr with diethyl ether-methanol-acetic acid (200:1:0.04, v/v/v). After UV detection of PGB~ (Rs = 0.48), the zone with R 10.50-0.70 was scraped off immediately and transferred to 25 ml of a mixture of diethyl ether-methanol (90: 10, v/v) at 0°. After mixing and centrifugation, the organic layer was evaporated under a stream of nitro-
86
ENZYMES A N D RECEPTORS" P U R I F I C A T I O N AND ASSAY
[13]
gen to 1 ml. After counting an aliquot of the solution, the total amount of PGH~ was calculated based on the known specific radioactivity. Then diethyl ether was added to give a stock solution of 200/zg of PGH~ per milliliter, which was stored at - 8 0 °. The yield was about 20%. Enzyme Assay. To 0.4 ml of incubation solution containing 0.1 M Tris, 0.1 M phosphate, 1 mM EDTA, 4 mM GSH, pH 7.4, an amount of PGE isomerase activity was added to get a conversion to PGE2 of between 30 and 70%. The reaction was started by the addition of radioactive PGH2 (2/zg, 5.7 nmol in 4/zl of ethanol). Incubation was carried out for exactly 1 min at 20°. The reaction was terminated by the rapid addition of 75/zl of 0.2 M citric acid, and then 1.2 ml of diethyl ether followed immediately by mixing and cooling at 0°. After centrifugation, the organic phase was separated and evaporated under a stream of nitrogen to approximately 25/A (not dry). After spotting as a 0.5-cm band on a 10 × 10 cm (0.1 mm thick) HPTLC plate (Merck) provided with a reference containing PGF2~, PGE2 and PGD2, the plate was developed immediately with diethyl ether-methanol-acetic acid (90: 2: 0.5, v/v/v). After drying in air, the plate was sprayed with phosphomolybdic acid in ethanol (100 g/liter) and heated. From the intensity of the blue spots, the conversion into PGE2 could be estimated. For quantitative determination, the blue spots of PGH, PGD, PGE, and PGF~ were scraped off (Re values 0.90, 0.55, 0.37, and 0.22, respectively) and the silica gel was transferred to a liquid scintillation vial to which 1.0 ml of methanol and 10 ml of toluene scintillator (Packard) were added. Radioactivity was measured with a liquid scintillation spectrometer. From the result, the amount of PGE2 formed was calculated. Calculation of Enzyme Activity. If the amount of PGE2 formed is plotted as a function of the amount of protein used, curves like the one in Fig.
PGE (nmol) 6
j o
fo---------o
/° I
o
5
J
lo protein (pg)
FIG. I. Productionof PGEs as a functionof the amount of protein in the 1.5% Triton supernatant. Assay methodas described in the text.
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
87
l are obtained. A linear relationship is found in a certain range of enzyme concentration and from the slope, the specific activity of the enzyme preparation tested (micromoles PGEz formed per minute per milligram of protein at 20°) can be calculated (duration of incubation is always 1 min). If the right amount of enzyme is taken, one or two incubations are sufficient to estimate the specific activity. The enzyme concentration should not be too low because then the accuracy is poor owing to the nonenzymic PGE2 formation. With excess enzyme a shortage of endoperoxide substrate prevents a reliable determination.
Purification P r o c e d u r e The results of the procedures used for the fractionation are summarized in the table. All manipulations were carried out between 0 ° and 4 °"
Step 1. 13,000 g Supernatant. Deep-frozen sheep seminal vesicles (225 g), trimmed free from fat and connective tissue, were homogenized in 75-g portions with 110 ml of 0.05 M Tris-HC1, pH 8.0, containing 10 mM EDTA disodium salt and 1 mM diethyldithiocarbamate using a Sorvall Omnimixer (twice for 30 sec) and a VirTis 45 homogenizer (3 x 10 sec). The homogenate was centrifuged for 20 min at 13,000 g. Step 2. Microsomes. The supernatant was filtered through two layers of cheesecloth and centrifuged for 45 min at 130,000 g. The pellets were rehomogenized, using a Potter-Elvehjem homogenizer, in 350 ml of 0.05 M Tris-HCl, pH 8.0, containing 0.1 M sodium perchlorate, 2 mM EDTA, and 0.5 mM diethyldithiocarbamate. Step 3. Perchlorate-Washed Microsomes. The homogenate was centrifuged again for 60 min at 130,000 g. The washed microsomes, consistPURIFICATION OF P G H - P G E ISOMERASE
Step 1. 2. 3. 4. 5. 6. 7. 8.
13,000 g Supernatant Microsomes Perchlorate-washed microsomes Tween-treated microsomes Washed Tween-treated microsomes Triton X-100 supernatant DEAE-ceUulose Hydroxyapatite-agarose (unbound protein fractions)
Volume (ml)
Protein (mg)
Specific activity (units)
450 420 210 140 140 140 30 45
8955 2860 1722 987 450 231 25 9
1.0 1.7 1.7 2.2 1.3 1.4 1.7 1.4
88
ENZYMES A N D RECEPTORS" PURIFICATION AND ASSAY
[13]
ing of a white fluffy layer and a red tightly packed pellet, were then homogenized in 180 ml of 0.05 M Tris-HCl, pH 8.0, containing 0.5 mM EDTA and 0.1 mM diethyl dithiocarbamate. Step 4. Tween-Treated Microsomes. Twenty-one milliliters of a 10% fresh aqueous Tween 20 (w/v) solution were added to the microsomal suspension to give a final concentration of I% Tween 20 in a total volume of 210 ml. The Tween 20 homogenate was centrifuged for 60 min at 130,000 g. The pellets were rehomogenized in 120 ml of 0.5 mM GSH, 0.5 mM EDTA adjusted with 1 M Na~CO3 to pH 8.0. Step 5. Washed Tween-Treated Microsomes. To the resuspended pellet 14 ml of a 10% Tween 20 solution were added, making a total volume of 140 ml. The homogenate was centrifuged for 60 min at 130,000 g. The pellets were homogenized in 0.5 mM GSH, 0.5 mM EDTA, pH 8, in a total volume of 120 ml. Step 6. 1.5% Triton Supernatant. By the addition of 21 ml of fresh aqueous 10% Triton X-100 (w/v, Packard) the Triton X-100 concentration was made 1.5%. The mixture was left for 30 min and then centrifuged for 60 min at 130,000 g. To the clear supernatant (112 ml), ethylene glycol (28 ml) was added to give a final concentration of 20% in a final volume of 140 ml. Step 7. Chromatography on DEAE-Cellulose. DEAE-cellulose (Whatman DE-52 microgranular) was treated as described in the Whatman Manual. The DEAE-cellulose column (1.6 x 70 cm) was equilibrated with a solution containing 0.5 mM EDTA, 0.5 mM GSH, 0.1 mM dithiothreitol (DTE, Merck), 0.5% Triton X-100 (w/v), 20% ethylene glycol (v/v) and adjusted to pH 8.0 with 1 M Na~CO3. After application of the Triton supematant, elution was continued first with 50 ml of equilibration buffer until the unbound protein had eluted and then with a linear gradient of 250 ml of equilibration buffer and 250 ml of equilibration buffer plus 0.1 M potassium phosphate, pH 8.0, made by an LKB Ultrograd, in 24 hr. The flow rate was about 18 ml/hr. The elution profile of this column is presented in Fig. 2. The fractions with a specific activity above 1.3 were combined (30 ml). Step 8. Chromatography on Hydroxyapatite-Agarose. A column (25 x 1.6 cm) was prepared of hydroxyapatite-agarose (LKB) and equilibrated with 30 mM potassium phosphate, pH 7.7, plus 0.1 mM EDTA, 0.1 mM DTE, 0.5 mM GSH, 0.5% Triton X-100, and 20% ethylene glycol. The pooled fractions of the DEAE chromatography were diluted with water made 0.5% in Triton X-100 and 20% in ethylene glycol to give a phosphate concentration of 30 raM. After application to the hydroxyapatite-agarose column and washing with 30 ml of starting buffer, the bound protein was eluted stepwise by increasing the phosphate concentration
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
89
specific activity
I
I
[ P043- ] (mot/t)
absorbance
//
0.4
///
0.3
protein (mg/ml)
0.075
//
0.050
1.0
0025
0.5
0.2 0.1 0
20
40
60
80 fraction number
F1G. 2. Elution pattern of the DEAE-celluiose column loaded with the Triton X-100 supernatant. Fractions of 20 rain were collected. , Absorbance at 280 nm; .... , concentration of PO4s- of the eluent; --., protein concentration. from 30 to 60 m M (step 1, 40 ml) and from 60 to 100 m M phosphate (step 2, 70 ml). The flow rate was about 15 ml/hr. Isomerase activity eluted in two parts: about half of the activity did not bind to the column and the other half eluted in step 1 with 60 m M phosphate. The specific activity o f the fractions containing the unbound protein was higher than that for the fractions eluted in step 1. H o w e v e r , the specific activity had increased only little with respect to the 13,000 g supernatant, probably because o f inactivation of the P G E isomerase during the purification procedure. Properties
Stability. The P G H - P G E isomerase is a highly unstable enzyme; it can be partially protected from inactivation during the purification procedure by addition o f thiol compounds in the buffers. In Fig. 3 the time-dependent inactivation at 0° is shown. The half-life o f the isomerase activity is about 20 hr at 0 °. This rapid inactivation o f the isomerase is one o f the main reasons why the specific activity does not increase (see the table). O t h e r factors, e.g., lipid requirement and influence of detergent, may also be responsible. A protein fraction o f the hydroxyapatite column showed the same lability, indicating that proteolytic digestion is probably not the reason. The inactivation can be suppressed by storage at - 8 0 °. Cofactor Requirement. P G H - P G E isomerase specifically needs gluta-
90
ENZYMES AND RECEPTORS; PURIFICATION AND ASSAY
[13]
enzyme (nmol/miactivity n) 40f~ 3.0
2.0 ©
1.0 0.5 0.4
0.3
°\
0.2
o
2',.
72 time
96
(hr)
FIG. 3. Time-dependent loss of isomerase activity in Triton X-100 supernatant at 0°. Assay with 8/~g of protein.
thione to exhibit its activity. However, there is no stoichiometric oxidation of glutathione. 5 Blocking reagents for SH groups, such as p-hydroxymercuribenzoate and N-ethylmaleimide, destroy the isomerase activity even if after incubation with these reagents an excess of glutathione is added. 6 pH Optimum. The P G H - P G E isomerase present in the Triton X-100 supernatant has a wide pH optimum between pH 5.5 and 7.0. At lower GSH concentrations (0.5 mM GSH), the optimum is sharper around pH 7.0.
Molecular Mass and Purity. The unbound hydroxyapatite column fractions show two protein bands with molecular mass between 60,000 and 67,000 daltons after SDS-slab gel electrophoresis on polyacrylamide in the presence of 2-mercaptoethanol (staining with Coomassie Brilliant 6 M. E. Gerritsen, T. P. Parks, and M. P. Printz, Biochim. Biophys. Acta 619, 196 (1980).
[14]
PGI~ SYNTHASE
91
Blue R250). The sensitive silver stain 7 indicated the presence in these fractions of some low molecular mass proteins (10,000 to 30,000), indicating that the purification procedure does not result in a completely homogeneous P G H - P G E isomerase. 7 B. R. Oakley, D. R. Kirsch, and N. R. Morris, Anal. Biochem. 105, 361 (1980),
[14] P r e p a r a t i o n
and Assay of Prostacyclin Synthase
By JOHN A. SALMON and ROOERICK J. FLOWER Prostacyclin is the most potent naturally occurring inhibitor of platelet aggregation and is also a powerful vasodilator. 1-3 It is formed by a rearrangement of the prostaglandin endoperoxide PGH~, the reaction being catalyzed by an enzyme commonly known as prostacyclin synthase (see Fig. 1). The enzyme is located in mammalian blood vessels, and although prostacyclin is generated by many organs it is often difficult to determine whether this is because all tissues contain vascular elements or whether there are genuine extravascular sources. It is the endothelium that contains the highest amounts of prostacyclin synthase per gram of tissue, but vascular smooth muscle can also synthesize substantial amounts of prostacyclin. Prostacyclin is unstable, with a half-life of approximately 10 min at physiological temperature and pH; it hydrolyzes quantitatively and nonenzymically to 6-keto-PGFl~ (see Fig. 1). Only bioassays permit the assay of prostacyclin itself in biological samples, and therefore the majority of investigators have sought to assess prostacyclin synthase by measuring the formation of 6-keto-PGFl~ by radiochemical, radioimmunoassay (RIA), or physicochemical procedures. Many of these assays are fully described elsewhere in this volume, and details will not be repeated here. Choice of Substrate Prostacyclin synthase converts prostaglandin endoperoxides to prostacyclin, and therefore the most convenient methods of assessing the enS. Moncada, R. J. Gryglewski, S. Bunting, and J. R. Vane, Nature (London) 263, 663 (1976). 2R. J. Gryglewski, S. Bunting, S. Moncada, R. J. Flower, and J.R. Vane, Prostaglandins 12, 685 (1976).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
ENZYMES AND RECEPTORS: PURIFICATION AND ASSAY
[13]
reaction. The optimum glutathione concentration lies between 10-4 and 10-3 M. Without glutathione, no enzymic PGD2 formation could be demonstrated. pH Optimum. Incubations were done between pH 4 and 9.4 in the presence of 1 mM glutathione. Optimum conversion was obtained between pH 7 and 8. 3 Inhibitory Substances. A number of compounds were tested that might inhibit the isomerization either by interference with the substrate binding site or with the glutathione binding site of the enzyme. Only those compounds that can react with free SH groups, such as 15-hydroperoxyarachidonic acid and p-hydroxymercuribenzoate, were found to inhibit the conversion. 3
[13] Purification
of PGH-PGE Isomerase Vesicular Glands
from Sheep
By P. MOONEN, M. BUYTENHEK, and D. H. NUGTEREN For studies on prostaglandin biosynthesis, the microsomal fraction of sheep vesicular glands is more or less the "classical" starting material. The biosynthesis of prostaglandin E (PGE) 1 from certain unsaturated fatty acids is a membrane-bound process that requires two enzymes acting sequentially. 2,a The first enzyme, which catalyzes the conversion of certain essential fatty acids (e.g., arachidonic acid) into prostaglandin H (PGH) is referred to as PGH synthase. This protein has been fully purified both from bovine and sheep vesicular gland microsomes. 4 The second enzyme needs glutathione (GSH) for its activity and isomerizes the endoperoxy group of PGH to a 9-keto-, 11-hydroxyl arrangement (PGE). This socalled prostaglandin endoperoxide-E isomerase ( P G H - P G E isomerase, EC 5.3.99.3) has been partially purified from bovine vesicular gland microsomes. 5 In this contribution our approaches to purification of P G H PGE isomerase from sheep vesicular gland microsomes are described.
i For nomenclature of prostaglandins see N. A. Nelson, J. Med. Chem. 17, 911 (1974). 2 D. H. Nugteren and E. Hazelhof, Biochim. Biophys. Acta 326, 448 (1973). 3 T. Miyamoto, S. Yamamoto, and O. Hayaishi, Proc. Natl. Acad. Sci. U.S.A. 71, 3645 (1974). 4 F. J. van der Ouderaraa and M. Buytenhek, this volume [9]. 5 N. Ogino, T. Miyamoto, S. Yamamoto, and O. Hayaishi, J. Biol. Chem. 252, 890 (1977).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
85
Assay Method Principle. The endoperoxide used as substrate in the assay is not stable in aqueous medium: it has a half-life of about 10 min at pH 7.4 and 20°. The main degradation products are PGE and PGD in a ratio of about 3 : 1. Therefore, incubations are carried out for only 1 min with different amounts of enzyme and with a fixed amount of 14C-labeled endoperoxide. After quick acidification and extraction with ether, the products formed and the remaining endoperoxide are rapidly separated by silica gelHPTLC. Quantification of the enzyme activity is possible by scraping off the radioactive bands, counting the radioactivity, and calculating the nanomoles of PGE formed. Preparation of Substrate (see also this volume [12]). Purified prostaglandin endoperoxide synthase4 (4.5 mg of protein, specific activity 16.9 U/mg) was preincubated with 1 mg of hemin in 16 ml of 0.1 M Tris-HCl, pH 8, for 5 min at 0°. [1-14C]Arachidonic acid (Radiochemical Centre, Amersham, 23 × 106 dpm) was added to unlabeled arachidonic acid to a total of 9.0 mg. The fatty acid plus 3 mg of hydroquinone were dissolved in a 250-ml Erlenmeyer flask in 40 ml of 0.1 M Tris-0.1 M phosphate, 1 mM EDTA, pH 8.0. The turbid suspension was carefully saturated with air and placed in a water bath at 29°. The enzyme solution was warmed up and subsequently transferred to the fatty acid suspension; the mixture was then incubated for 2.5 min at 29°. The Erlenmeyer flask was shaken manually, and a stream of air was introduced into the flask. The reaction was terminated by pouring the mixture rapidly into 18 ml of 0.2 M citric acid plus 100 ml of ethyl acetate cooled in ethanol-solid CO2, immediately followed by thoroughly shaking. The reaction vessel was cooled so that the water phase just did not freeze. After separation of the two phases, the ethyl acetate phase was immediately decanted and the water phase was rapidly extracted with 25 ml of cooled ethyl acetate. The combined ethyl acetate phases were cooled to - 80° for 4 hr to freeze out the water, and then filtered at - 80° over a cooled glass filter G3 (15-40/zm). The precipitate was washed with 25 ml of cooled ethyl acetate. The filtered ethyl acetate was evaporated under reduced pressure to approximately 1 ml and spotted in a cold room as a 10-cm band on a 20 × 20 cm 0.5 mm-thick silica gel 60 F254 plate (Merck); 10/zg of prostaglandin B~ or B2 had been spotted beforehand as a reference. The plate was developed without delay at 4° for 1 hr with diethyl ether-methanol-acetic acid (200:1:0.04, v/v/v). After UV detection of PGB~ (Rs = 0.48), the zone with R 10.50-0.70 was scraped off immediately and transferred to 25 ml of a mixture of diethyl ether-methanol (90: 10, v/v) at 0°. After mixing and centrifugation, the organic layer was evaporated under a stream of nitro-
86
ENZYMES A N D RECEPTORS" P U R I F I C A T I O N AND ASSAY
[13]
gen to 1 ml. After counting an aliquot of the solution, the total amount of PGH~ was calculated based on the known specific radioactivity. Then diethyl ether was added to give a stock solution of 200/zg of PGH~ per milliliter, which was stored at - 8 0 °. The yield was about 20%. Enzyme Assay. To 0.4 ml of incubation solution containing 0.1 M Tris, 0.1 M phosphate, 1 mM EDTA, 4 mM GSH, pH 7.4, an amount of PGE isomerase activity was added to get a conversion to PGE2 of between 30 and 70%. The reaction was started by the addition of radioactive PGH2 (2/zg, 5.7 nmol in 4/zl of ethanol). Incubation was carried out for exactly 1 min at 20°. The reaction was terminated by the rapid addition of 75/zl of 0.2 M citric acid, and then 1.2 ml of diethyl ether followed immediately by mixing and cooling at 0°. After centrifugation, the organic phase was separated and evaporated under a stream of nitrogen to approximately 25/A (not dry). After spotting as a 0.5-cm band on a 10 × 10 cm (0.1 mm thick) HPTLC plate (Merck) provided with a reference containing PGF2~, PGE2 and PGD2, the plate was developed immediately with diethyl ether-methanol-acetic acid (90: 2: 0.5, v/v/v). After drying in air, the plate was sprayed with phosphomolybdic acid in ethanol (100 g/liter) and heated. From the intensity of the blue spots, the conversion into PGE2 could be estimated. For quantitative determination, the blue spots of PGH, PGD, PGE, and PGF~ were scraped off (Re values 0.90, 0.55, 0.37, and 0.22, respectively) and the silica gel was transferred to a liquid scintillation vial to which 1.0 ml of methanol and 10 ml of toluene scintillator (Packard) were added. Radioactivity was measured with a liquid scintillation spectrometer. From the result, the amount of PGE2 formed was calculated. Calculation of Enzyme Activity. If the amount of PGE2 formed is plotted as a function of the amount of protein used, curves like the one in Fig.
PGE (nmol) 6
j o
fo---------o
/° I
o
5
J
lo protein (pg)
FIG. I. Productionof PGEs as a functionof the amount of protein in the 1.5% Triton supernatant. Assay methodas described in the text.
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
87
l are obtained. A linear relationship is found in a certain range of enzyme concentration and from the slope, the specific activity of the enzyme preparation tested (micromoles PGEz formed per minute per milligram of protein at 20°) can be calculated (duration of incubation is always 1 min). If the right amount of enzyme is taken, one or two incubations are sufficient to estimate the specific activity. The enzyme concentration should not be too low because then the accuracy is poor owing to the nonenzymic PGE2 formation. With excess enzyme a shortage of endoperoxide substrate prevents a reliable determination.
Purification P r o c e d u r e The results of the procedures used for the fractionation are summarized in the table. All manipulations were carried out between 0 ° and 4 °"
Step 1. 13,000 g Supernatant. Deep-frozen sheep seminal vesicles (225 g), trimmed free from fat and connective tissue, were homogenized in 75-g portions with 110 ml of 0.05 M Tris-HC1, pH 8.0, containing 10 mM EDTA disodium salt and 1 mM diethyldithiocarbamate using a Sorvall Omnimixer (twice for 30 sec) and a VirTis 45 homogenizer (3 x 10 sec). The homogenate was centrifuged for 20 min at 13,000 g. Step 2. Microsomes. The supernatant was filtered through two layers of cheesecloth and centrifuged for 45 min at 130,000 g. The pellets were rehomogenized, using a Potter-Elvehjem homogenizer, in 350 ml of 0.05 M Tris-HCl, pH 8.0, containing 0.1 M sodium perchlorate, 2 mM EDTA, and 0.5 mM diethyldithiocarbamate. Step 3. Perchlorate-Washed Microsomes. The homogenate was centrifuged again for 60 min at 130,000 g. The washed microsomes, consistPURIFICATION OF P G H - P G E ISOMERASE
Step 1. 2. 3. 4. 5. 6. 7. 8.
13,000 g Supernatant Microsomes Perchlorate-washed microsomes Tween-treated microsomes Washed Tween-treated microsomes Triton X-100 supernatant DEAE-ceUulose Hydroxyapatite-agarose (unbound protein fractions)
Volume (ml)
Protein (mg)
Specific activity (units)
450 420 210 140 140 140 30 45
8955 2860 1722 987 450 231 25 9
1.0 1.7 1.7 2.2 1.3 1.4 1.7 1.4
88
ENZYMES A N D RECEPTORS" PURIFICATION AND ASSAY
[13]
ing of a white fluffy layer and a red tightly packed pellet, were then homogenized in 180 ml of 0.05 M Tris-HCl, pH 8.0, containing 0.5 mM EDTA and 0.1 mM diethyl dithiocarbamate. Step 4. Tween-Treated Microsomes. Twenty-one milliliters of a 10% fresh aqueous Tween 20 (w/v) solution were added to the microsomal suspension to give a final concentration of I% Tween 20 in a total volume of 210 ml. The Tween 20 homogenate was centrifuged for 60 min at 130,000 g. The pellets were rehomogenized in 120 ml of 0.5 mM GSH, 0.5 mM EDTA adjusted with 1 M Na~CO3 to pH 8.0. Step 5. Washed Tween-Treated Microsomes. To the resuspended pellet 14 ml of a 10% Tween 20 solution were added, making a total volume of 140 ml. The homogenate was centrifuged for 60 min at 130,000 g. The pellets were homogenized in 0.5 mM GSH, 0.5 mM EDTA, pH 8, in a total volume of 120 ml. Step 6. 1.5% Triton Supernatant. By the addition of 21 ml of fresh aqueous 10% Triton X-100 (w/v, Packard) the Triton X-100 concentration was made 1.5%. The mixture was left for 30 min and then centrifuged for 60 min at 130,000 g. To the clear supernatant (112 ml), ethylene glycol (28 ml) was added to give a final concentration of 20% in a final volume of 140 ml. Step 7. Chromatography on DEAE-Cellulose. DEAE-cellulose (Whatman DE-52 microgranular) was treated as described in the Whatman Manual. The DEAE-cellulose column (1.6 x 70 cm) was equilibrated with a solution containing 0.5 mM EDTA, 0.5 mM GSH, 0.1 mM dithiothreitol (DTE, Merck), 0.5% Triton X-100 (w/v), 20% ethylene glycol (v/v) and adjusted to pH 8.0 with 1 M Na~CO3. After application of the Triton supematant, elution was continued first with 50 ml of equilibration buffer until the unbound protein had eluted and then with a linear gradient of 250 ml of equilibration buffer and 250 ml of equilibration buffer plus 0.1 M potassium phosphate, pH 8.0, made by an LKB Ultrograd, in 24 hr. The flow rate was about 18 ml/hr. The elution profile of this column is presented in Fig. 2. The fractions with a specific activity above 1.3 were combined (30 ml). Step 8. Chromatography on Hydroxyapatite-Agarose. A column (25 x 1.6 cm) was prepared of hydroxyapatite-agarose (LKB) and equilibrated with 30 mM potassium phosphate, pH 7.7, plus 0.1 mM EDTA, 0.1 mM DTE, 0.5 mM GSH, 0.5% Triton X-100, and 20% ethylene glycol. The pooled fractions of the DEAE chromatography were diluted with water made 0.5% in Triton X-100 and 20% in ethylene glycol to give a phosphate concentration of 30 raM. After application to the hydroxyapatite-agarose column and washing with 30 ml of starting buffer, the bound protein was eluted stepwise by increasing the phosphate concentration
[13]
PGH-PGE
ISOMERASE FROM SHEEP GLANDS
89
specific activity
I
I
[ P043- ] (mot/t)
absorbance
//
0.4
///
0.3
protein (mg/ml)
0.075
//
0.050
1.0
0025
0.5
0.2 0.1 0
20
40
60
80 fraction number
F1G. 2. Elution pattern of the DEAE-celluiose column loaded with the Triton X-100 supernatant. Fractions of 20 rain were collected. , Absorbance at 280 nm; .... , concentration of PO4s- of the eluent; --., protein concentration. from 30 to 60 m M (step 1, 40 ml) and from 60 to 100 m M phosphate (step 2, 70 ml). The flow rate was about 15 ml/hr. Isomerase activity eluted in two parts: about half of the activity did not bind to the column and the other half eluted in step 1 with 60 m M phosphate. The specific activity o f the fractions containing the unbound protein was higher than that for the fractions eluted in step 1. H o w e v e r , the specific activity had increased only little with respect to the 13,000 g supernatant, probably because o f inactivation of the P G E isomerase during the purification procedure. Properties
Stability. The P G H - P G E isomerase is a highly unstable enzyme; it can be partially protected from inactivation during the purification procedure by addition o f thiol compounds in the buffers. In Fig. 3 the time-dependent inactivation at 0° is shown. The half-life o f the isomerase activity is about 20 hr at 0 °. This rapid inactivation o f the isomerase is one o f the main reasons why the specific activity does not increase (see the table). O t h e r factors, e.g., lipid requirement and influence of detergent, may also be responsible. A protein fraction o f the hydroxyapatite column showed the same lability, indicating that proteolytic digestion is probably not the reason. The inactivation can be suppressed by storage at - 8 0 °. Cofactor Requirement. P G H - P G E isomerase specifically needs gluta-
90
ENZYMES AND RECEPTORS; PURIFICATION AND ASSAY
[13]
enzyme (nmol/miactivity n) 40f~ 3.0
2.0 ©
1.0 0.5 0.4
0.3
°\
0.2
o
2',.
72 time
96
(hr)
FIG. 3. Time-dependent loss of isomerase activity in Triton X-100 supernatant at 0°. Assay with 8/~g of protein.
thione to exhibit its activity. However, there is no stoichiometric oxidation of glutathione. 5 Blocking reagents for SH groups, such as p-hydroxymercuribenzoate and N-ethylmaleimide, destroy the isomerase activity even if after incubation with these reagents an excess of glutathione is added. 6 pH Optimum. The P G H - P G E isomerase present in the Triton X-100 supernatant has a wide pH optimum between pH 5.5 and 7.0. At lower GSH concentrations (0.5 mM GSH), the optimum is sharper around pH 7.0.
Molecular Mass and Purity. The unbound hydroxyapatite column fractions show two protein bands with molecular mass between 60,000 and 67,000 daltons after SDS-slab gel electrophoresis on polyacrylamide in the presence of 2-mercaptoethanol (staining with Coomassie Brilliant 6 M. E. Gerritsen, T. P. Parks, and M. P. Printz, Biochim. Biophys. Acta 619, 196 (1980).
[14]
PGI~ SYNTHASE
91
Blue R250). The sensitive silver stain 7 indicated the presence in these fractions of some low molecular mass proteins (10,000 to 30,000), indicating that the purification procedure does not result in a completely homogeneous P G H - P G E isomerase. 7 B. R. Oakley, D. R. Kirsch, and N. R. Morris, Anal. Biochem. 105, 361 (1980),
[14] P r e p a r a t i o n
and Assay of Prostacyclin Synthase
By JOHN A. SALMON and ROOERICK J. FLOWER Prostacyclin is the most potent naturally occurring inhibitor of platelet aggregation and is also a powerful vasodilator. 1-3 It is formed by a rearrangement of the prostaglandin endoperoxide PGH~, the reaction being catalyzed by an enzyme commonly known as prostacyclin synthase (see Fig. 1). The enzyme is located in mammalian blood vessels, and although prostacyclin is generated by many organs it is often difficult to determine whether this is because all tissues contain vascular elements or whether there are genuine extravascular sources. It is the endothelium that contains the highest amounts of prostacyclin synthase per gram of tissue, but vascular smooth muscle can also synthesize substantial amounts of prostacyclin. Prostacyclin is unstable, with a half-life of approximately 10 min at physiological temperature and pH; it hydrolyzes quantitatively and nonenzymically to 6-keto-PGFl~ (see Fig. 1). Only bioassays permit the assay of prostacyclin itself in biological samples, and therefore the majority of investigators have sought to assess prostacyclin synthase by measuring the formation of 6-keto-PGFl~ by radiochemical, radioimmunoassay (RIA), or physicochemical procedures. Many of these assays are fully described elsewhere in this volume, and details will not be repeated here. Choice of Substrate Prostacyclin synthase converts prostaglandin endoperoxides to prostacyclin, and therefore the most convenient methods of assessing the enS. Moncada, R. J. Gryglewski, S. Bunting, and J. R. Vane, Nature (London) 263, 663 (1976). 2R. J. Gryglewski, S. Bunting, S. Moncada, R. J. Flower, and J.R. Vane, Prostaglandins 12, 685 (1976).
METHODS IN ENZYMOLOGY, VOL. 86
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181986-8