29
Biochimica et Biophysics Acta, 572 (1979) 29-42 0 ElsevierlNorth-Holland Biomedical Press
BBA 57298
ON THE HAEMOPROTEIN CHARACTER OF PROSTAGLANDIN ENDOPEROXIDE SYNTHETASE F.J. VAN DER OUDERAA, M. BUYTENHEK, F.J. SLIKKERVEER Unilever Research
Vlaardingen,
P.O.Box
114, 3130 AC Vlaardingen
and D.A. VAN DORP (The Netherlands)
(Received June 19th, 1978) Key words: Prostaglandin Prostaglandin biosynthesis
endoperoxidase
synthetase;
Hemoprotein
characterization;
Summary Prostaglandin endoperoxide synthetase is a membrane-bound glycoprotein isolated as a dimer of molecular weight of approximately 129 000 consisting of two identical polypeptide chains. Several research workers have reported that haemin (ferri-protoporphyrinIX) is required to restore the full enzymic activity of the pure apoprotein. Difference spectroscopy shows association of haemin up to two molecules per polypeptide chain of molecular weight 70 000. Both the cyclooxygenase and the peroxidase activity displayed by the enzyme can be optimally stimulated by similar quantities of haemin. The restored haemin-enzyme complex has a millimolar absorption coefficient at 408 nm of 61 mM_l - cm-l per haem. Using this value, the presence of non-haem iron in the enzyme is virtually excluded. These findings and the spectra of the reassociated enzyme-haemin complex point to a haemoprotein character. The availability of haemin to the enzyme ‘might play a regulating role in its action. Introduction Isolation of homogeneous prostaglandin endoperoxide synthetase has recently been described [l-4] and its substrate specificity has been studied extensively [ 5-71. The enzyme catalyses two concerted reactions resulting in the conversion of, for instance, the essential fatty acid arachidonic acid to the prostaglandin endoperoxide PGHz. We have shown that this enzyme is a membrane-bound glycoprotein. From data obtained by analytical ultracentrifugation and gel chromatography, we concluded that the protein is isolated as a dimer in solutions of 0.1% Tween-20 [3]. Furthermore, values of approx. 70 000 have been reported for the molecular weight of the polypeptide chains using acrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulphate [2-41. Roth et al. [4] have recently demonstrated that the protein consists of identical polypeptide chains.
30
The pure enzyme displays both cyclooxygenase and peroxidase activities. Haemin is necessary for the oxygenase activity [1,3,8] and stimulates the peroxidase activity [ 31. We have postulated that haemin acts as an easily dissociable prosthetic group [ 31. Enzyme prepared according to our procedure [3] contains only traces of metals. Hemler et al. [Z] have reported that the enzyme contains both haem and non-haem iron, although no conclusive quantitative data were provided. In a more recent paper, Hemler and Lands [9] suggest a tetrameric enzyme structure of an AzB2 type, containing two molecules of haem and two atoms of nonhaem iron per tetramer. In this paper, we present spectral and additional kinetic evidence that the enzyme must be regarded as a haemoprotein devoid of non-haem iron. Binding of two molecules of haemin per polypeptide chain (2 mol haemin per 70 000 g enzyme) was observed. A thorough reinvestigation confirmed our earlier findings that no appreciable quantity of non-haem iron is present in this enzyme. Materials and methods Materials. Ultrodex, Ultrogel and Ampholine were purchased from LKB. Pure soyabean Iipoxygenase-1 was a gift from Dr. Spaapen, Laboratory of Organic Chemistry, Utrecht. Catalase was obtained from Boehringer and bovine serum albumin from Miles. Fatty acids were from our laboratory stocks. 13-Hydroperoxylinoleic acid was prepared from linoleic acid by incubation with soyabean lipoxygenase from Sigma [lo] ; the reaction product was purified by thin-layer chromatography. Concentrations of linoleic acid hydroperoxide were determined from the absorbance at 234 nm, using a millimolar absorption coefficient of 25 000 mM-1 s cm-” ]ll]_ Octylglucoside [12] was prepared by Mr. Mank of our laboratory using a Koenings-Knorr synthesis as modified by Schroeder and Green [13] ; the synthesized material was purified by thin-layer chromatography (purity: >95%; m.p.: 68-75°C) and its purity was confirmed by NMR and mass spectroscopy. Octaethyleneglycol dodecyl ether was obtained from Nikko Chemicals, Tokyo. Enzyme isolation. Procedures for the isolation of prostaglandin endoperoxide synthetase from sheep vesicular glands are described in the literature [l-3], The enzyme preparation used for the present studies was obtained by the following modified procedure (only the major deviations from the original procedure [3] are described here). Fresh seminal vesicles (250 g) were collected in the slaughterhouse and a microsomal preparation was made. Perchloratewashed microsomes were solubilised using 1% Tween-20; the 200 000 X g supernatant of this fraction was applied to a column of UItrogel ACA 34 (65 X 5 cm). The oxygenase-containing fractions from this column were concentrated and the concentrate was applied to a slab gel (10.4 X 24 cm) of Ultrodex containing 0.1% Tween-20, 10% glycerol and 2% Ampholine, pH 5-7. Slab gel isoelectric focusing was carried out at 0°C using a LKB Multiphor apparatus with a ‘constant-power’ supply. After 16 h, the current stabilised at 2 mA at 1500 A. Thirty fractions were scraped off the plate and assayed for oxygenase activity. Ultrodex was removed by centrifugation and the enzyme material eoncen-
31
trated. The concentrate was applied to a column of Ultrogel ACA 34 (60 X 1.5 cm) eluted with a buffer containing 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 0.01% sodium azide, 0.1 mM EDTA amd 0.1% octylglucoside. In this way, Tween-20 bound to the enzyme is replaced by the readily dialysable non-ionic detergent octylglucoside. Fractions of the eluate were monitored for oxygenase activity and for protein and iron content. A yield of 60 mg of homogenous enzyme was obtained with a specific activity of 14 ymol eicosatrienoic acid converted per mg protein per min at 25°C. The concentrated eluate showed no appreciable decrease in activity after 2 months’ storage at 0°C. Protein de termination. Protein concentrations were measured according to Lowry et al. [14]. To each sample 20 ~1 of 10% sodium dodecyl sulphate was added to prevent turbidity caused by nonionic detergent. Bovine serum albumin served as a reference. It was dissolved with the same buffer as used for eluting the columns. Haemin determination. Absolute amounts of haemin were determined as dipyridyl haemochromes according to the procedure of Falke [ 151. Iron determination. The protein samples were analysed for iron by graphite furnace atomic absorption spectrometry using a Perkin-Elmer model 400 atomic absorption spectrometer equipped with a Perkin-Elmer HGA-72 graphite furnace atomizer and an atomization control unit. Correction for nonatomic absorption signals was made with a background corrector. The atomizer was programmed as follows: drying for 30 s at 100°C; programmed heating from 100 to 1100°C in 60 s; ashing for 30 s at 1100°C; atomizing for 15 s at 2650°C. Concentrations of up to 0.10 pg iron per ml were measured at 248.3 nm. To avoid dilution errors, concentrations of 0.10-1.00 pg iron per ml were measured at 372.0 nm. The iron standard solution ‘Titrisol’ (FeC13 in dilute hydrochloric acid) was used to prepare the standard solutions and these as well as the samples were stored in polyethylene bottles. The elution buffer of the ACA 34 column contained 0.02 pg iron per ml. Equilibrium ultrafiltration. Equilibrium ultrafiltration was carried out in an Amicon model 52 ultrafiltration cell with an XM-50 filter. The enzyme was equilibrated with haemin solutions until no further increase in absorbance between two successive filtrations was observed. Ultraviolet-visible spectroscopy. All spectroscopic measurements were performed with a Varian Cary 219-UV-visible spectrometer. For difference spectroscopy tandem cells, pathlength 0.438 cm per compartment, were used. Spectra of the reduced protein were measured using Thunberg cuvettes in an argon atmosphere. Determination of enzymic activity. Determination of the oxygenase and peroxidase activities of the enzyme was carried out essentially as described previously [ 31. Results Iron analyses Fig. 1 shows the protein and the iron content of prostaglandin endoperoxide synthetase and of three reference proteins after elution from a column of Ultrogel ACA 34. The cyclooxygenase activity of prostaglandin endoperoxide
oxygenase (25t)l funitslml) 50
10
40
08
30
0.6
20
0.4
10
0.2
pmstaglandin endoperoxide synthetase
50
60
70
80
i 90
100 etution volume/ml
Fig. I. Gel chromatography of prostaglandin endoperoxide synthetase and of the reference proteins. Protein content ( C-----O), iron content (0 -D) and cyclooxygenase activity (a ------A) were determined In fractions from an Ultrogel ACA 34 column. The column was eiuted at 4’C with a buffer containing 50 mM Trls-HCl (pH 8). 0.1% octylglucoside, 0.1 M N&l. 0.01% sodium azide and 0.1 mM EDTA. The buffer flow was 8 ml/h. Interpolation of the observed partition coefficient (0.41) for catake in a calibration graph for ACA 34 indicates that this protein behaves as a dimer. Interpolation using the Same graph gave a molecular weight of 155 000 for the octylgtucoside-prosta~~d~ endoperoxide complex.
synthetase is also shown in Fig. 1. The reference proteins were catalase, a a non-haem iron protein and bovine serum haemoprotein, lipoxygenase, albumin, which latter contains no iron. EDTA (0.1 mM) was added to the elution buffer to avoid nonspecific iron complex formation by the proteins. In all experiments the protein and iron peaks of the iron-containing proteins were in alignment. The quantitative results of the iron analyses of the column fractions are summarised in Table I. For catalase, the amount of iron obtained by atomic absorption (0.7 atom iron per subunit of 62 000) is in reasonable agreement with the amount of haemin bound to that enzyme (0.6 molecule haemin per subunit of 62 000) determined from the absorbance at 408 nm (c = 115 rnM_l-
33
TABLE
I
IRON CONTENT THETASE (PGES) The ACA 34 elution
(PER POLYPEPTIDE AND OF REFERENCE buffer contained
Preparation
PGES 1 PGES 2 PGES 3 Bovine serum albumin Cat&se Lipoxygenase
0.02
Iron */atom
0.14 0.31 0.19
Biochimica et Biophysics Acta, 572 (1979) 29-42 0 ElsevierlNorth-Holland Biomedical Press
BBA 57298
ON THE HAEMOPROTEIN CHARACTER OF PROSTAGLANDIN ENDOPEROXIDE SYNTHETASE F.J. VAN DER OUDERAA, M. BUYTENHEK, F.J. SLIKKERVEER Unilever Research
Vlaardingen,
P.O.Box
114, 3130 AC Vlaardingen
and D.A. VAN DORP (The Netherlands)
(Received June 19th, 1978) Key words: Prostaglandin Prostaglandin biosynthesis
endoperoxidase
synthetase;
Hemoprotein
characterization;
Summary Prostaglandin endoperoxide synthetase is a membrane-bound glycoprotein isolated as a dimer of molecular weight of approximately 129 000 consisting of two identical polypeptide chains. Several research workers have reported that haemin (ferri-protoporphyrinIX) is required to restore the full enzymic activity of the pure apoprotein. Difference spectroscopy shows association of haemin up to two molecules per polypeptide chain of molecular weight 70 000. Both the cyclooxygenase and the peroxidase activity displayed by the enzyme can be optimally stimulated by similar quantities of haemin. The restored haemin-enzyme complex has a millimolar absorption coefficient at 408 nm of 61 mM_l - cm-l per haem. Using this value, the presence of non-haem iron in the enzyme is virtually excluded. These findings and the spectra of the reassociated enzyme-haemin complex point to a haemoprotein character. The availability of haemin to the enzyme ‘might play a regulating role in its action. Introduction Isolation of homogeneous prostaglandin endoperoxide synthetase has recently been described [l-4] and its substrate specificity has been studied extensively [ 5-71. The enzyme catalyses two concerted reactions resulting in the conversion of, for instance, the essential fatty acid arachidonic acid to the prostaglandin endoperoxide PGHz. We have shown that this enzyme is a membrane-bound glycoprotein. From data obtained by analytical ultracentrifugation and gel chromatography, we concluded that the protein is isolated as a dimer in solutions of 0.1% Tween-20 [3]. Furthermore, values of approx. 70 000 have been reported for the molecular weight of the polypeptide chains using acrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulphate [2-41. Roth et al. [4] have recently demonstrated that the protein consists of identical polypeptide chains.
30
The pure enzyme displays both cyclooxygenase and peroxidase activities. Haemin is necessary for the oxygenase activity [1,3,8] and stimulates the peroxidase activity [ 31. We have postulated that haemin acts as an easily dissociable prosthetic group [ 31. Enzyme prepared according to our procedure [3] contains only traces of metals. Hemler et al. [Z] have reported that the enzyme contains both haem and non-haem iron, although no conclusive quantitative data were provided. In a more recent paper, Hemler and Lands [9] suggest a tetrameric enzyme structure of an AzB2 type, containing two molecules of haem and two atoms of nonhaem iron per tetramer. In this paper, we present spectral and additional kinetic evidence that the enzyme must be regarded as a haemoprotein devoid of non-haem iron. Binding of two molecules of haemin per polypeptide chain (2 mol haemin per 70 000 g enzyme) was observed. A thorough reinvestigation confirmed our earlier findings that no appreciable quantity of non-haem iron is present in this enzyme. Materials and methods Materials. Ultrodex, Ultrogel and Ampholine were purchased from LKB. Pure soyabean Iipoxygenase-1 was a gift from Dr. Spaapen, Laboratory of Organic Chemistry, Utrecht. Catalase was obtained from Boehringer and bovine serum albumin from Miles. Fatty acids were from our laboratory stocks. 13-Hydroperoxylinoleic acid was prepared from linoleic acid by incubation with soyabean lipoxygenase from Sigma [lo] ; the reaction product was purified by thin-layer chromatography. Concentrations of linoleic acid hydroperoxide were determined from the absorbance at 234 nm, using a millimolar absorption coefficient of 25 000 mM-1 s cm-” ]ll]_ Octylglucoside [12] was prepared by Mr. Mank of our laboratory using a Koenings-Knorr synthesis as modified by Schroeder and Green [13] ; the synthesized material was purified by thin-layer chromatography (purity: >95%; m.p.: 68-75°C) and its purity was confirmed by NMR and mass spectroscopy. Octaethyleneglycol dodecyl ether was obtained from Nikko Chemicals, Tokyo. Enzyme isolation. Procedures for the isolation of prostaglandin endoperoxide synthetase from sheep vesicular glands are described in the literature [l-3], The enzyme preparation used for the present studies was obtained by the following modified procedure (only the major deviations from the original procedure [3] are described here). Fresh seminal vesicles (250 g) were collected in the slaughterhouse and a microsomal preparation was made. Perchloratewashed microsomes were solubilised using 1% Tween-20; the 200 000 X g supernatant of this fraction was applied to a column of UItrogel ACA 34 (65 X 5 cm). The oxygenase-containing fractions from this column were concentrated and the concentrate was applied to a slab gel (10.4 X 24 cm) of Ultrodex containing 0.1% Tween-20, 10% glycerol and 2% Ampholine, pH 5-7. Slab gel isoelectric focusing was carried out at 0°C using a LKB Multiphor apparatus with a ‘constant-power’ supply. After 16 h, the current stabilised at 2 mA at 1500 A. Thirty fractions were scraped off the plate and assayed for oxygenase activity. Ultrodex was removed by centrifugation and the enzyme material eoncen-
31
trated. The concentrate was applied to a column of Ultrogel ACA 34 (60 X 1.5 cm) eluted with a buffer containing 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 0.01% sodium azide, 0.1 mM EDTA amd 0.1% octylglucoside. In this way, Tween-20 bound to the enzyme is replaced by the readily dialysable non-ionic detergent octylglucoside. Fractions of the eluate were monitored for oxygenase activity and for protein and iron content. A yield of 60 mg of homogenous enzyme was obtained with a specific activity of 14 ymol eicosatrienoic acid converted per mg protein per min at 25°C. The concentrated eluate showed no appreciable decrease in activity after 2 months’ storage at 0°C. Protein de termination. Protein concentrations were measured according to Lowry et al. [14]. To each sample 20 ~1 of 10% sodium dodecyl sulphate was added to prevent turbidity caused by nonionic detergent. Bovine serum albumin served as a reference. It was dissolved with the same buffer as used for eluting the columns. Haemin determination. Absolute amounts of haemin were determined as dipyridyl haemochromes according to the procedure of Falke [ 151. Iron determination. The protein samples were analysed for iron by graphite furnace atomic absorption spectrometry using a Perkin-Elmer model 400 atomic absorption spectrometer equipped with a Perkin-Elmer HGA-72 graphite furnace atomizer and an atomization control unit. Correction for nonatomic absorption signals was made with a background corrector. The atomizer was programmed as follows: drying for 30 s at 100°C; programmed heating from 100 to 1100°C in 60 s; ashing for 30 s at 1100°C; atomizing for 15 s at 2650°C. Concentrations of up to 0.10 pg iron per ml were measured at 248.3 nm. To avoid dilution errors, concentrations of 0.10-1.00 pg iron per ml were measured at 372.0 nm. The iron standard solution ‘Titrisol’ (FeC13 in dilute hydrochloric acid) was used to prepare the standard solutions and these as well as the samples were stored in polyethylene bottles. The elution buffer of the ACA 34 column contained 0.02 pg iron per ml. Equilibrium ultrafiltration. Equilibrium ultrafiltration was carried out in an Amicon model 52 ultrafiltration cell with an XM-50 filter. The enzyme was equilibrated with haemin solutions until no further increase in absorbance between two successive filtrations was observed. Ultraviolet-visible spectroscopy. All spectroscopic measurements were performed with a Varian Cary 219-UV-visible spectrometer. For difference spectroscopy tandem cells, pathlength 0.438 cm per compartment, were used. Spectra of the reduced protein were measured using Thunberg cuvettes in an argon atmosphere. Determination of enzymic activity. Determination of the oxygenase and peroxidase activities of the enzyme was carried out essentially as described previously [ 31. Results Iron analyses Fig. 1 shows the protein and the iron content of prostaglandin endoperoxide synthetase and of three reference proteins after elution from a column of Ultrogel ACA 34. The cyclooxygenase activity of prostaglandin endoperoxide
oxygenase (25t)l funitslml) 50
10
40
08
30
0.6
20
0.4
10
0.2
pmstaglandin endoperoxide synthetase
50
60
70
80
i 90
100 etution volume/ml
Fig. I. Gel chromatography of prostaglandin endoperoxide synthetase and of the reference proteins. Protein content ( C-----O), iron content (0 -D) and cyclooxygenase activity (a ------A) were determined In fractions from an Ultrogel ACA 34 column. The column was eiuted at 4’C with a buffer containing 50 mM Trls-HCl (pH 8). 0.1% octylglucoside, 0.1 M N&l. 0.01% sodium azide and 0.1 mM EDTA. The buffer flow was 8 ml/h. Interpolation of the observed partition coefficient (0.41) for catake in a calibration graph for ACA 34 indicates that this protein behaves as a dimer. Interpolation using the Same graph gave a molecular weight of 155 000 for the octylgtucoside-prosta~~d~ endoperoxide complex.
synthetase is also shown in Fig. 1. The reference proteins were catalase, a a non-haem iron protein and bovine serum haemoprotein, lipoxygenase, albumin, which latter contains no iron. EDTA (0.1 mM) was added to the elution buffer to avoid nonspecific iron complex formation by the proteins. In all experiments the protein and iron peaks of the iron-containing proteins were in alignment. The quantitative results of the iron analyses of the column fractions are summarised in Table I. For catalase, the amount of iron obtained by atomic absorption (0.7 atom iron per subunit of 62 000) is in reasonable agreement with the amount of haemin bound to that enzyme (0.6 molecule haemin per subunit of 62 000) determined from the absorbance at 408 nm (c = 115 rnM_l-
33
TABLE
I
IRON CONTENT THETASE (PGES) The ACA 34 elution
(PER POLYPEPTIDE AND OF REFERENCE buffer contained
Preparation
PGES 1 PGES 2 PGES 3 Bovine serum albumin Cat&se Lipoxygenase
0.02
Iron */atom
0.14 0.31 0.19
