Biochimica et Biophysica Acta, 1160 (1992) 269-274 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
269
BBAPRO 34331
Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii Miguel Aguilar, Jacobo Cfirdenas and Emilio Fernfindez Departamento de Bioqu[mica, Biologla Molecular y Fisiolog[a, Facultad de Ciencias, Universidad de Cdrdoba, Cdrdoba (Spain) (Received 6 March 1992)
Key words: Molybdenumcofactor; Molybdopterin;Molybdoenzyme;(C. reinhardtii) A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120°C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway. Introduction
All known eukaryotic molyboenzymes contain a very similar, if not identical, molybdenum cofactor [1,2]. In its active form, MoCo is a very labile chemical species highly sensitive to oxygen [3] and, thus, its direct analysis has been impossible to perform until now. First approaches to the MoCo structure were based on the fact that aerobic denaturing of MoCo yielded a fluorescent signal typical of pterins [3]. Several MoCoderived fluorescent species have been characterized: form A, form B, urothione and a blue-fluorescence compound [4-6]. Chemical and spectroscopic characterization of these species and of alkylated molybdopterin has decisively contributed to the current understanding of MoCo structure [7] that consists of a 6-alkyl pterin, with a four-carbon side-chain containing l ' - e n e , l ' , 2 '-dithiol,3 ',4'-dihydroxyl, 4 ' - p h o s p h a t e groups. MoCo oxidation products can be used as an indirect method to determine MoCo concentration [1]. This procedure avoids the shortcomings of the active MoCo instability and the many factors affecting the usual method for MoCo assay by complementation with extracts from the Neurospora crassa nit-1 mutant [1,8].
Correspondence to: E. Fermlndez, Departamento de Bioquimica, Biologla Molecular y Fisiologia, Facultad de Ciencias, Universidad de C6rdoba, 14071 C6rdoba, Spain. Abbreviations: MoCo, molybdenum cofactor; MoCo-FOP, MoCo fluorescent oxidation product; SO, sulfite oxidase; XO, xanthine oxidase; XDH, xanthine dehydrogenase.
Although form B species has a structure containing more atoms from intact MoCo than form A, this latter species obtained after oxidation of MoCo with iodine and dephosphorylation with alkaline phosphatase has been suggested to be a more suitable form for MoCo assay by HPLC [1]. In this work, we report on a reproducible and quantitative procedure,gnuch faster and easier than others previously r e p o r t d d to obtain a fluorescent species derived from MoCo oxidation and on a method for determination of this oxidized species by reverse-phase HPLC. The method is applied to assay either active or inactive MoCo in crude extracts from wild-type and MoCo-deficient mutant strains from Chlamydomonas reinhardtii and the results suggest that, even in the absence of active MoCo, feed-back inhibition of its biosynthetic pathway is taking place. Materials and Methods
Organisms and growth conditions. Wild-type strain 6145c from Chlamydomonas reinhardtii and mutant strains 203 (nit-2), 104 (nit-4), 102 (nit-5, nit-6), 307 (nit-3) and F23 (nit-7) have been previously characterized [9-13]. The nit-1 mutant strain from Neurospora crassa was supplied by the Fungal Genetics Stock Center (Arcata, USA). Chlamydomonas reinhardtii cells were grown at 25°C under continuous and saturating illumination conditions, either in a liquid minimum medium with CO 2enriched air (5%, v / v ) or in a solid TAP medium (Tris-acetate-phosphate), containing 10 mM ammonium chloride as the sole nitrogen source [12]. The N.
270
crassa nit-1 mutant was grown aerobically in an orbital shaker at 27°C in the Fries basal medium containing 10 mM ammonium chloride as nitrogen source [14]. After three days of growth in the dark, mycelia were collected by filtration, thoroughly washed with distilled water and transferred to the basal medium containing 8 mM potassium nitrate to induce synthesis of aponitrate reductase. After 5 h, mycelia were collected, washed with distilled water and frozen at - 4 0 ° C until use. Preparation of extracts. C. reinhardtii cells were broken by thawing frozen cell pellets in 50 mM potassium phosphate buffer (pH 7.0). When required, buffer was previously degassed with O2-free Ar and the extraction procedure was performed under a gas phase of Ar. The nit-1 mutant mycelia were broken with acid-washed sand (1.7 g per g of mycelium) in an ice-cold mortar. The resulting mixture was extracted in buffer A (25 mM potassium phosphate buffer (pH 7.5), containing 25 mM sodium molybdate and 1 mM EDTA). The suspension was centrifuged at 27000 x g for 10 min and the supernatant was immediately used for MoCo assays. Active MoCo determination by complementation. Active MoCo was assayed by determining reconstituted NR activity in a mixture of 200 /zl of extracts of nit-1 mutant from N. crassa and 100 ~1 of a source of MoCo preincubated under optimal complementation conditions at 15°C during 1 h [15]. Reconstituted NR activity was determined after adding 300/xl of the NR activity assay mixture to the complementation mixture and incubating during 10 min at 25°C as previously described [15], by measuring the nitrite formed spectrophotometrically [16]. 1 unit of active MoCo is defined as the amount of MoCo that yields 1 unit of reconstituted NR activity expressed as the amount of enzyme which catalyzes the reduction of 1 /xmol of nitrate per min. Obtention of the phosphorylated and dephosphorylated MoCo oxidation product. Algal extracts and enzyme preparations used as MoCo sources were obtained in a 50 mM potassium phosphate buffer (pH 7.0). The MoCo oxidation product was obtained by autoclaving samples at 120°C during 20 min. Autoclaved samples, always protected from light, were centrifuged in Eppendorf tubes at 12000 rpm for 15 min and the resulting supernatant was immediately analysed by HPLC. Dephosphorylation of the MoCo oxidation product was carried out by incubating samples with 0.2 mg of calf intestinal alkaline phosphatase per ml during 16 h in 25 mM Tris-HC1 buffer (pH 8.0), containing 20 mM MgCI 2. Thin-layer chromatography. Thin-layer chromatography was carried out on plates (5 × 20 cm) of Silica Gel 60 from Merck. Samples of 5/xl were applied and run using a 3% NH4C1 solution in water as eluant.
Reverse-phase HPLC. The MoCo oxidation products were applied to a reverse-phase HPLC column Spheri-5 ODS (220 x 4.6 mm, 5 Izm) from Brownlee, by using a Rheodyne injector equipped with a 200 Izl loop. The column was previously equilibrated with 10 mM potassium phosphate buffer (pH 7.0). Samples were eluted with a continuous gradient 0 - 3 0 % (v/v) methanol in the same phosphate buffer during 30 rain with a flow rate of 1 ml/min. A characteristic fluorescence signal of pterins at 440-460 nm was detected with a Schoeffel FS970 spectrofluorimetric detector by using an excitation wavelength of 390 nm and an emission filter of 418 nm. The column was thoroughly washed with pure methanol until no signal eluted and then it was reequilibrated with 10 mM potassium phosphate buffer (pH 7.0). Purification of the MoCo-FOP. The MoCo oxidation product was purified from SO, XO or C. reinhardtii extracts according to the following protocol: (a) The enzyme preparation in 10 mM ammonium acetate (pH 6.5), was autoclaved at 120°C for 20 min in Eppendorf tubes and then centrifuged at 15000 rpm. (b), The resulting supernatant was passed through a hydroxyapatite column (0.5 x 2 cm) equilibrated with 10 mM ammonium acetate buffer (pH 6.5). The column was washed with 5 ml of the buffer and the MoCo oxidation product was eluted with 50 mM potassium phosphate buffer (pH 7.0). (c), The solution from (b) was chromatographed through a reverse-phase HPLC column as detailed above and the peak fraction eluting at t r 10 min was collected. Fluorescence spectra. The fluorescence spectra of purified MoCo-FOP in 10 mM potassium phosphate buffer (pH 7.0) were recorded in a spectrofluorometer Perkin Elmer MPS 43A. Enzymes and chemicals. Homogeneous C. reinhardtii xanthine dehydrogenase (1000 U / m g protein, kindly provided by Drs. J. Alamillo and M. Pineda), commercial milk xanthine oxidase (Grade I, 0.59 U / m g protein) and chicken-liver sulfite oxidase (70 U / r a g protein) were used as sources of MoCo. Calf intestinal mucose alkaline phosphatase type I-S (10 U / r a g protein) was used for dephosphorylation experiments. Enzymes were from Sigma, St. Louis, MO, USA. All other chemicals were from Sigma or Merck, Darmstadt, Germany. Results
MoCo extracted from different sources is a very unstable species easily oxidizable in air, that yields several oxidation products [4]. By autoclaving enzyme preparations of XO and SO at 120°C for 20 min, a blue fluorescence during UV light exposure appeared, typical of pterins. Molybdopterin from MoCo, both active and inactive, present in XO or SO, has been quanti-
271
m C) z w (D (/3 w rY 0
E D
[3 L L-
0-
20.0
v
10.0
0
269
BBAPRO 34331
Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii Miguel Aguilar, Jacobo Cfirdenas and Emilio Fernfindez Departamento de Bioqu[mica, Biologla Molecular y Fisiolog[a, Facultad de Ciencias, Universidad de Cdrdoba, Cdrdoba (Spain) (Received 6 March 1992)
Key words: Molybdenumcofactor; Molybdopterin;Molybdoenzyme;(C. reinhardtii) A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120°C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway. Introduction
All known eukaryotic molyboenzymes contain a very similar, if not identical, molybdenum cofactor [1,2]. In its active form, MoCo is a very labile chemical species highly sensitive to oxygen [3] and, thus, its direct analysis has been impossible to perform until now. First approaches to the MoCo structure were based on the fact that aerobic denaturing of MoCo yielded a fluorescent signal typical of pterins [3]. Several MoCoderived fluorescent species have been characterized: form A, form B, urothione and a blue-fluorescence compound [4-6]. Chemical and spectroscopic characterization of these species and of alkylated molybdopterin has decisively contributed to the current understanding of MoCo structure [7] that consists of a 6-alkyl pterin, with a four-carbon side-chain containing l ' - e n e , l ' , 2 '-dithiol,3 ',4'-dihydroxyl, 4 ' - p h o s p h a t e groups. MoCo oxidation products can be used as an indirect method to determine MoCo concentration [1]. This procedure avoids the shortcomings of the active MoCo instability and the many factors affecting the usual method for MoCo assay by complementation with extracts from the Neurospora crassa nit-1 mutant [1,8].
Correspondence to: E. Fermlndez, Departamento de Bioquimica, Biologla Molecular y Fisiologia, Facultad de Ciencias, Universidad de C6rdoba, 14071 C6rdoba, Spain. Abbreviations: MoCo, molybdenum cofactor; MoCo-FOP, MoCo fluorescent oxidation product; SO, sulfite oxidase; XO, xanthine oxidase; XDH, xanthine dehydrogenase.
Although form B species has a structure containing more atoms from intact MoCo than form A, this latter species obtained after oxidation of MoCo with iodine and dephosphorylation with alkaline phosphatase has been suggested to be a more suitable form for MoCo assay by HPLC [1]. In this work, we report on a reproducible and quantitative procedure,gnuch faster and easier than others previously r e p o r t d d to obtain a fluorescent species derived from MoCo oxidation and on a method for determination of this oxidized species by reverse-phase HPLC. The method is applied to assay either active or inactive MoCo in crude extracts from wild-type and MoCo-deficient mutant strains from Chlamydomonas reinhardtii and the results suggest that, even in the absence of active MoCo, feed-back inhibition of its biosynthetic pathway is taking place. Materials and Methods
Organisms and growth conditions. Wild-type strain 6145c from Chlamydomonas reinhardtii and mutant strains 203 (nit-2), 104 (nit-4), 102 (nit-5, nit-6), 307 (nit-3) and F23 (nit-7) have been previously characterized [9-13]. The nit-1 mutant strain from Neurospora crassa was supplied by the Fungal Genetics Stock Center (Arcata, USA). Chlamydomonas reinhardtii cells were grown at 25°C under continuous and saturating illumination conditions, either in a liquid minimum medium with CO 2enriched air (5%, v / v ) or in a solid TAP medium (Tris-acetate-phosphate), containing 10 mM ammonium chloride as the sole nitrogen source [12]. The N.
270
crassa nit-1 mutant was grown aerobically in an orbital shaker at 27°C in the Fries basal medium containing 10 mM ammonium chloride as nitrogen source [14]. After three days of growth in the dark, mycelia were collected by filtration, thoroughly washed with distilled water and transferred to the basal medium containing 8 mM potassium nitrate to induce synthesis of aponitrate reductase. After 5 h, mycelia were collected, washed with distilled water and frozen at - 4 0 ° C until use. Preparation of extracts. C. reinhardtii cells were broken by thawing frozen cell pellets in 50 mM potassium phosphate buffer (pH 7.0). When required, buffer was previously degassed with O2-free Ar and the extraction procedure was performed under a gas phase of Ar. The nit-1 mutant mycelia were broken with acid-washed sand (1.7 g per g of mycelium) in an ice-cold mortar. The resulting mixture was extracted in buffer A (25 mM potassium phosphate buffer (pH 7.5), containing 25 mM sodium molybdate and 1 mM EDTA). The suspension was centrifuged at 27000 x g for 10 min and the supernatant was immediately used for MoCo assays. Active MoCo determination by complementation. Active MoCo was assayed by determining reconstituted NR activity in a mixture of 200 /zl of extracts of nit-1 mutant from N. crassa and 100 ~1 of a source of MoCo preincubated under optimal complementation conditions at 15°C during 1 h [15]. Reconstituted NR activity was determined after adding 300/xl of the NR activity assay mixture to the complementation mixture and incubating during 10 min at 25°C as previously described [15], by measuring the nitrite formed spectrophotometrically [16]. 1 unit of active MoCo is defined as the amount of MoCo that yields 1 unit of reconstituted NR activity expressed as the amount of enzyme which catalyzes the reduction of 1 /xmol of nitrate per min. Obtention of the phosphorylated and dephosphorylated MoCo oxidation product. Algal extracts and enzyme preparations used as MoCo sources were obtained in a 50 mM potassium phosphate buffer (pH 7.0). The MoCo oxidation product was obtained by autoclaving samples at 120°C during 20 min. Autoclaved samples, always protected from light, were centrifuged in Eppendorf tubes at 12000 rpm for 15 min and the resulting supernatant was immediately analysed by HPLC. Dephosphorylation of the MoCo oxidation product was carried out by incubating samples with 0.2 mg of calf intestinal alkaline phosphatase per ml during 16 h in 25 mM Tris-HC1 buffer (pH 8.0), containing 20 mM MgCI 2. Thin-layer chromatography. Thin-layer chromatography was carried out on plates (5 × 20 cm) of Silica Gel 60 from Merck. Samples of 5/xl were applied and run using a 3% NH4C1 solution in water as eluant.
Reverse-phase HPLC. The MoCo oxidation products were applied to a reverse-phase HPLC column Spheri-5 ODS (220 x 4.6 mm, 5 Izm) from Brownlee, by using a Rheodyne injector equipped with a 200 Izl loop. The column was previously equilibrated with 10 mM potassium phosphate buffer (pH 7.0). Samples were eluted with a continuous gradient 0 - 3 0 % (v/v) methanol in the same phosphate buffer during 30 rain with a flow rate of 1 ml/min. A characteristic fluorescence signal of pterins at 440-460 nm was detected with a Schoeffel FS970 spectrofluorimetric detector by using an excitation wavelength of 390 nm and an emission filter of 418 nm. The column was thoroughly washed with pure methanol until no signal eluted and then it was reequilibrated with 10 mM potassium phosphate buffer (pH 7.0). Purification of the MoCo-FOP. The MoCo oxidation product was purified from SO, XO or C. reinhardtii extracts according to the following protocol: (a) The enzyme preparation in 10 mM ammonium acetate (pH 6.5), was autoclaved at 120°C for 20 min in Eppendorf tubes and then centrifuged at 15000 rpm. (b), The resulting supernatant was passed through a hydroxyapatite column (0.5 x 2 cm) equilibrated with 10 mM ammonium acetate buffer (pH 6.5). The column was washed with 5 ml of the buffer and the MoCo oxidation product was eluted with 50 mM potassium phosphate buffer (pH 7.0). (c), The solution from (b) was chromatographed through a reverse-phase HPLC column as detailed above and the peak fraction eluting at t r 10 min was collected. Fluorescence spectra. The fluorescence spectra of purified MoCo-FOP in 10 mM potassium phosphate buffer (pH 7.0) were recorded in a spectrofluorometer Perkin Elmer MPS 43A. Enzymes and chemicals. Homogeneous C. reinhardtii xanthine dehydrogenase (1000 U / m g protein, kindly provided by Drs. J. Alamillo and M. Pineda), commercial milk xanthine oxidase (Grade I, 0.59 U / m g protein) and chicken-liver sulfite oxidase (70 U / r a g protein) were used as sources of MoCo. Calf intestinal mucose alkaline phosphatase type I-S (10 U / r a g protein) was used for dephosphorylation experiments. Enzymes were from Sigma, St. Louis, MO, USA. All other chemicals were from Sigma or Merck, Darmstadt, Germany. Results
MoCo extracted from different sources is a very unstable species easily oxidizable in air, that yields several oxidation products [4]. By autoclaving enzyme preparations of XO and SO at 120°C for 20 min, a blue fluorescence during UV light exposure appeared, typical of pterins. Molybdopterin from MoCo, both active and inactive, present in XO or SO, has been quanti-
271
m C) z w (D (/3 w rY 0
E D
[3 L L-
0-
20.0
v
10.0
0
