Isolation and partial structure determination of a clonidine-displacing substance from bovine lung and brain

Journal of the Autonomic Nervous System 72 Ž1998. 86–93

Isolation and partial structure determination of a clonidine-displacing substance from bovine lung and brain Matthew Grigg a , Ian F. Musgrave b, Colin J. Barrow a

a,)

Department of Chemistry, The UniÕersity of Melbourne, ParkÕille, Victoria 3052, Australia b Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Abstract A large scale extraction and isolation method was developed for the purification of clonidine-displacing substance ŽCDS. activity from bovine lung or brain. This optimised method used direct freeze drying of tissue, hexane removal of lipids, and methanol extraction of CDS activity. Using a bioassay directed isolation strategy a new CDS compound was purified from an extract of bovine lung. The isolation strategy involved subsequent steps of flash C-18 chromatography, ion exchange, size exclusion, and C-18 HPLC. An HPLC detection method was developed and applied to show that the new CDS is present in both lung and brain tissue. Spectroscopic data for this new CDS indicates that it is related to guanosine, but is not noradrenaline, adrenaline, histamine, agmatine, guanosine, GMP, GDP or GTP. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Imidazoline; CDS; Clonidine; Isolation; Lung; Brain

1. Introduction The anti-hypertensive drug clonidine defines distinct, non-adrenergic receptors or sites which recognise its imidazoline structure ŽBousquet et al., 1992; De Vos et al., 1994; Ernsberger et al., 1995a.. These sites are called imidazoline receptors and appear to be of physiological importance ŽBousquet et al., 1992; De Vos et al., 1994; Ernsberger et al., 1995a.. Two distinct types of imidazoline receptors exits, the I 1 sites which have high affinity for clonidine ŽErnsberger et al., 1988; Michel and Ernsberger, 1992., and I 2 sites which have high affinity for idazoxan ŽTesson et al., 1991.. Other sites, including an I 3 site may also exist ŽKing et al., 1995; Molderings et al., 1995.. Imidazoline receptors have been implicated in multiple physiological functions, including the hypertension produced by clonidine and other imidazolines ŽGomez et al., 1991; Mermet and Quintin, 1991; Tibirica et al., 1991; Atlas et al., 1992; Campbell and Potter, 1994; Smyth et al., 1995; Glavin and Smyth, 1995.. The discovery of imidazoline receptors came about partly due to the discovery in 1984 of an endogenous compound present in both bovine and rat brain which ) Corresponding author. Fax: [email protected]

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competes for w3 Hx-clonidine binding to a 2-adrenergic receptors ŽAtlas et al., 1987.. The structure of this compound, labelled ‘clonidine-displacing substance’ ŽCDS., has not been determined, although indications are that the compound is a heat stable, methanol soluble, non-peptidic, small molecule ŽAtlas et al., 1987; Synestos et al., 1991; Goldberg-Stern et al., 1993.. Although CDS has been partially purified from human plasma ŽSmyth et al., 1995., human cerebrospinal fluid ŽSingh et al., 1995. and bovine lung ŽReis et al., 1992., its structure and biological activities remain elusive. There has been considerable literature disagreement on both the physicochemical and biological properties of CDS, possibly because of the range of extraction procedures, assay systems, and purification strategies employed by various groups ŽDiamant and Atlas, 1986; Atlas et al., 1987; Ernsberger et al., 1988; Synestos et al., 1991; Reis et al., 1992; Piletz et al., 1995.. Inconsistencies in properties such as CDS chromatographic elution behaviour ŽAtlas and Burnstein, 1984; Atlas et al., 1987., and stability ŽAtlas and Burnstein, 1984; Meeley et al., 1986., indicate that multiple CDS compounds are present in the tissue. Varying biological activity profiles for CDS also supports multiple CDS compounds, although some of these activities could be due to unrelated contaminants in the semipurified mixtures used for assay. Not until purified CDS is

0165-1838r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 9 2 - 7

M. Grigg et al.r Journal of the Autonomic NerÕous System 72 (1998) 86–93

isolated and structurally characterised will the biological properties of endogenous CDS compounds be unambiguously determined. Recently, agmatine, the decarboxylation product of arginine, was isolated as the first endogenous CDS compound from bovine brain ŽLoring, 1990; Li et al., 1994.. Although agmatine does not appear to have all the biological properties attributed to CDS, it clearly binds to imidazoline receptors and has some functional activity ŽLoring, 1990; Li et al., 1994.. In a recent study, agmatine levels in a variety of rat tissues were measured and agmatine was shown to be present in multiple tissue types including stomach, lung and brain ŽMeeley et al., 1986.. Singh et al. Ž1995. recently reported the presence of a non-catecholamine CDS in methanolic extracts of bovine lung, in a three-fold higher concentration than that found in bovine brain. In addition, agmatine does not have the physiochemical properties first reported for CDS ŽAtlas and Burnstein, 1984.. Therefore, there is at least one other CDS present in mammalian tissue. The aims of the study reported here were to: develop a large scale extraction method to overcome the difficulties in obtaining enough CDS material for structure determination; determine if CDS activity in lung is due to the same compound that is responsible for CDS activity in brain; develop an HPLC detection method for CDS; and to obtain structural information for a CDS other than agmatine.

2. Materials and methods 2.1. General Bovine lung and brain were obtained directly from Ralphs Abattoir ŽFootscray, Victoria., transported on ice and either snap frozen in liquid nitrogen and stored at y708C or immediately chopped and lyophilised or blended. All tissue homogenisation was performed in a Semak 5 l blender with variable setting on high. Centrifugation of homogenates was carried out in a Beckman J-21 centrifuge in 200 ml polypropylene containers. C-18 material used for flash columns was obtained from J.T. Baker ŽCA, USA.. Dowex 50-X8 cation exchange resin was obtained from BDH Chemicals ŽPoole, England.. Amberlite IRA 400 anion exchange resin was obtained from Hopkin & Williams ŽCA, USA.. Biogel P2 size exclusion resin Ž40–90 mm mesh. was obtained from Biorad Laboratories ŽCA, USA.. Column flow rates were kept constant using Econoglass columns connected to a Biotechnica peristaltic pump. Reverse phase high pressure liquid chromatography ŽHPLC. were performed using a Waters system with dual 510 pumps and a model 996 photodiode array detector controlled using Millenium software. Semi-preparative HPLC was carried out using a YMC RP-18 column Ž10

87

mm = 250 mm. with a flow rate of 3 mlrmin. All solvents were HPLC grade. Proton Ž1 H.-NMR spectra were recorded on a Bruker500 MHz NMR-spectrometer equipped with a microprobe. Chemical shifts are recorded as d values in parts per million Žppm.. w3 HxClonidine Ž61.9 Cirmmol. was obtained from New England Nuclear ŽMelbourne, Australia.. Other chemicals, including adrenaline, agmatine, clonidine, histamine, noradrenaline, phenylmethylsulphonyl fluoride, naphazoline, guanosine, GMP, GDP and GTP were obtained from Sigma ŽSydney, Australia.. 2.2. Extractions 2.2.1. Method 1 Fresh bovine lung and brain Ž200 g each. was extracted using the method of Meeley et al. Ž1986. to yield crude lung Ž1.0% yield. and brain extracts Ž2.0% yield.. Each extract was preabsorbed onto 10–15 g of C-18 resin and added to the head of a flash column Ž28 mm = 300 mm. containing 75 g of C-18 resin. Fractions were eluted with 200 ml water ŽFraction 1., 80:20 waterrmethanol ŽFraction 2., 50:50 waterrmethanol ŽFraction 3., and 80:20 methanolrdichloromethane ŽFraction 4.. 2.2.2. Method 2 Fresh bovine lung or brain Ž200 g each. was chopped into 3–5 cm3 pieces, blended in 3 vrw of distilled water, boiled for 5 min and centrifuged at 10 000 = g for 30 min at 08C. The supernatant was decanted, frozen and then lyophilised overnight. This was then extracted twice with methanol and dried in vacuo to yield crude lung Ž1.1% yield. and brain extracts Ž2.7% yield.. These extracts were chromatographed as above. 2.2.3. Method 3 Wet bovine lung and brain Ž200 g each. were blended in methanol Ž3 vrw., the solid was resuspended in methanol Ž2 vrw., sonicated, filtered, and the solvent removed in vacuo to yield crude lung Ž0.5% yield. and brain extracts Ž1.0% yield.. These extracts were chromatographed as above. 2.2.4. Method 4 Wet bovine lung and brain Ž200 g., were diced, frozen in liquid nitrogen, lyophilised, blended in methanol Ž3 vrw., filtered and dried in vacuo to yield crude lung Ž0.9% yield. and brain extracts Ž0.9% yield.. These extracts were chromatographed as above. 2.2.5. Method 5 Wet bovine lung Ž200 g., was diced, frozen in liquid nitrogen, lyophilised, blended in hexane Ž3 vrw., centrifuged, the supernatant dried and blended in methanol

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Ž3 vrw., filtered and dried in vacuo to yield crude lung Ž1.79% yield.. These extracts were chromatographed as above. 2.3. Large scale isolation of CDS actiÕity 2.3.1. Cation exchange chromatography Lung material extracted and chromatographed using extraction Method 5 was subjected to cation exchange chromatography on a Dowex 50-X8 column Ž37 g, 10 = 500 mm.. After resin acidification with 1 M HCl and washing with water, 132 mg of C-18 eluted material Ž45 CDS units. was dissolved in methanol, loaded on the column and eluted with water. The eluted material was lyophilised to provide a solid Ž21% yield by weight, 8% yield by activity.. 2.3.2. Anion exchange chromatography Lung material extracted and chromatographed using Method 5 was subjected to anion exchange chromatography on an Amberlite IRA-400 column Ž30 g, 10 = 500 mm.. After washing with water, 117 mg of fraction 2 Ž65 units of CDS. was dissolved in methanol, loaded and eluted with water. The eluted material was lyophilised to provide a solid Ž44% yield by weight, 65% yield by activity.. 2.3.3. Size exclusion chromatography Lung material obtained from an anion exchange column was dissolved in methanol and passed through a Biogel P2 column Ž103 g, 110 cm = 28 mm., eluting with water at 0.5 mlrmin. Twenty-four 1-h fractions were collected and dried in vacuo. 2.3.4. High pressure liquid chromatography (HPLC) Material was sequentially injected onto a YMC semipreparative C-18 column Ž7.5 = 250 mm. eluting with water containing 0.1% trifluoroacetic acid at a flow rate of 3 mlrmin. Biological activity eluted consistently with a UV peak at 21 min. The compound eluting at 21 min was partially characterised: UV-max, 280 nm; MS 489 or 275 ŽMHq. ; 1 H-NMR Ž500 MHz, d 6-DMSO. d 8.2 Žs, 1H, heteroaromatic.; d 5.8 Žs, 1H, anomeric.; d 5.4 Žs, 1H, OH.; d 5.2 Žs, 1H, OH.; d 5.1 Žs, 1H, OH., d 4.1 Žs, 1H, methine.; d 4.0 Žs, 1H, methine.; d 3.9 Žs, 1H, methine.; d 3.6 Ždd, 2H, methylene.. 2.3.5. HPLC comparison of lung and brain Extracts of both lung and brain obtained using extraction Method 4 were dissolved in 50% methanol and eluted from a semi-preparative C-18 HPLC column, using the method described above. Fractions were collected and bioassayed. In both cases, CDS activity corresponded with a UV-peak at 21 min, with photodiode array UV-spectra showing a 280-nm maxima.

2.3.6. HPLC analysis of standards Noradrenaline, adrenaline, agmatine, histamine, guanosine, GMP, GDP and GTP was injected onto a C-18 column and eluted using the standard method. Retention times and UV-spectra were compared with an injection of purified CDS material obtained from lung tissue. 2.4. Biological actiÕity 2.4.1. Membrane preparation Membranes were prepared from rat cerebral cortex by a modification of the method of Anis et al. Ž1990.. Rat cerebral cortex was homogenised in ice-cold buffer Ž30 vrw sucrose 0.32 M, HEPES 20 mM, EGTA 5 mM, phenylmethylsulphonyl fluoride 100 mM, sodium azide 1%, pH 7.4.. The homogenate was centrifuged for 10 min at 1000 = g then the supernatant was centrifuged at 15 000 = g for 30 min. The pellet resuspended in the sucrose buffer, and centrifuged again at 15 000 = g. The resulting pellet was then resuspended in a total of 48 ml wash buffer containing HEPES 5 mM and EDTA 0.5 mM, pH 7.4. This suspension was centrifuged twice at 48 000 = g, allowed to stand for 1 h at 48C to allow endogenous ligands to dissociate, centrifuged and resuspended in 12 ml of wash buffer. Then, 1 ml Aliquot’s were snap frozen in liquid nitrogen and stored at y708C for not more than 3 months before use. Protein concentrations were determined using the Coomassie Blue method ŽGogstad and Krutnes, 1982.. 2.4.2. Ligand binding assays CDS activity was measured by displacement of w3 Hxclonidine binding from membranes prepared from rat cerebral cortex using a modification of the method of Ernsberger et al. Ž1995b.. Briefly, an aliquot of membranes Žfinal concentration 0.2 mgrml. was incubated in polypropylene tubes in 200 ml of binding buffer with a final concentration of HEPES 5 mM, EGTA 0.5 mM, EDTA 0.5 mM, MgCl 2 0.5 mM, BSA 1% wrv, adjusted to pH 7.7 with TRIS base. Tubes were preincubated for 3 min at 258C and binding initiated by addition of w3 Hxclonidine Ž5 mM.. After incubation for 30 min, the reaction was terminated by rapid filtration onto Whatman GFrC glass filters presoaked in 1% high molecular weight polyethylene imine ŽAldrich, Australia. in a Brandel Harvester ŽBrandel, USA., and quickly washing the membranes with a 6-s wash of ice-cold buffer ŽHEPES 5 mM, EDTA 0.5 mM, pH 7.7.. Binding was estimated by liquid scintillation counting in a scintillation cocktail. Nonspecific binding was determined with naphazoline Ž100 mM.. The concentration of CDS activity was defined in CDS units where 1 unit of CDS activity was the amount required to displace 50% of bound w3 Hxclonidine in the assay system. Assays were carried out with 1r50 dilutions in methanol which did not affect binding. IC 50 , K d and

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Bmax values were obtained from logistic or hyperbolic curves fitted to the data using non-linear regression methods. K i values were obtained from IC 50 values by the method of Cheng and Prussoff Ž1973..

3. Results and discussion 3.1. DeÕelopment of a large scale extraction method Scheme 1. Isolation strategy for CDS from both bovine lung and brain.

We initially partially purified CDS from both bovine brain and lung according to the extraction method of Meeley et al. Ž1986. ŽMethod 1.. This standard extraction method was difficult to scale up to kilogram quantities because: Ža. initial homogenisation was time consuming; Žb. filtration of the homogenate was difficult; and Žc. large quantities of aqueous filtrate needed to be lyophilised. In addition, the water extraction step exposed CDS to active enzymes and the use of boiling water could degrade heat sensitive CDS molecules. Our initial modification of the standard method replaced time consuming filtration with centrifugation ŽMethod 2.. To eliminate heating, water extraction and the use of a homogeniser, we blended wet tissue directly in methanol and filtered ŽMethod 3.. However, scale up was difficult because removal of methanolrwater from the wet tissue extract using rotary evaporation was accompanied by foaming and was therefore slow. Foaming was eliminated by freeze drying wet tissue before methanol extraction ŽMethod 4.. A final modification involved a hexane extraction step after tissue lyophilisation and before methanol extraction, to remove inert fats ŽMethod 5.. Method 5 was effective for extracting CDS activity ŽTable 1. and

Method

Tissue

Extract yield Ž%.

CDS unitsrmg extract F1 F2 F3

F4

1

Lung Brain Lung Brain Lung Brain Lung Brain Lung

0.99 1.98 1.08 2.70 0.47 0.97 0.90 0.92 1.79

43.8 17.0 10.2 10.5 6.5 13.8 73.2 53.8 51.8

38.0 0 23.0 0 53.8 0 8.3 0 39.2

3 4 5

3.2. CDS isolation strategy Reverse-phase flash C-18 chromatography was chosen for the first purification step after extraction because of ease of scale-up. In addition, the majority of CDS activity was found to be retained after elution with water, so that cations responsible for nonspecific activity were readily removed ŽScheme 1.. Because the CDS molecule may be charged ŽAtlas and Burnstein, 1984. we incorporated an ion-exchange chromatography step directly after C-18 flash chromatography. CDS activity was retained on a cation exchange column and enriched when eluting off an anion exchange column ŽFig. 1., indicating that CDS is positively charged. An anion exchange step was therefore used to further purify CDS. Subsequent size exclusion chromatography gave a peak of CDS activity in fractions 8, 9 and 10 ŽFig. 2.. Fractions 8–10 were combined and further purified using C-18 HPLC. 3.3. DeÕelopment of an HPLC detection method for CDS

Table 1 CDS recovery from five extraction methods

2

amenable to scale up for use with kilogram quantities of either bovine lung or brain.

41.0 12.5 3.5 8.8 11.8 11.3 71.3 8.8 61.0

70.0 28.8 69.8 37.8 31.0 34.3 49.0 37.8 4.0

Extraction yield refers to percent wet weight recovered after extraction and before C-18 chromatography. Fractions were eluted from flash C-18 chromatography using: 100% water ŽF1., 20% methanol ŽF2.; 50% methanol ŽF3.; and 100% methanol ŽF4.. Based on polarity, cations responsible for non-specific activity should be in Fraction 1. CDS units were defined such that 1 unit of CDS activity was the amount required to displace 50% of bound w3 Hxclonidine in the assay system.

Although several gradients were applied to the HPLC separation of CDS from both lung and brain, the best

Fig. 1. Ion exchange behaviour of CDS after partial purification using C-18 chromatography. Cation exchange ŽDowex 50W-X8. retained most CDS activity, while anion exchange ŽAmberlite IRA400. enriched CDS activity by selectively retaining inactive anionic compounds.

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HPLC peak ŽFig. 3.. Analysis of the UV-spectrum of this HPLC peak, using a photodiode array detector, indicated the presence of a single compound. Approximately 50 mg of this compound was obtained and subjected to spectroscopic analysis. The developed HPLC detection method using photodiode-array detection was applied to initial extracts of bovine lung and brain. Although incompletely resolved, a peak with the correct UV-spectrum and retention time was observed in both brain and lung extracts. Semi-preparative Fig. 2. CDS activity in fractions eluted from a Biogel P2 size exclusion column over a 24-h period at a flow rate of 0.5 mlrmin.

separation was obtained using isocratic elution with 0.1% trifluoroacetic acid in water from a C-18 reverse-phase column. The presence of acid was necessary to obtain good resolution of CDS activity. The presence of organic solvents such as acetonitrile or methanol at levels down to 1% caused CDS activity to elute with the solvent front. Using a semi-preparative C-18 column the majority of CDS activity from Biogel fractions 8–10 eluted as a single

Fig. 3. ŽA. Semi-preparative HPLC isolation of CDS. Fractions 1–5 eluted isocratically in 0.1% TFA in water over 30 min and fractions 6–10 eluted in a gradient of 100%, 0.1% TFA in water to 100% methanol over 20 min. ŽB. CDS activity for each HPLC fraction. Fraction 4 contained a single compound for which spectroscopic data was obtained.

Fig. 4. ŽA. CDS activity for HPLC fractions from bovine brain Žopen bars. and lung Žhatched bars. extracts, eluted under isocratic conditions using 0.1% TFA. ŽB. HPLC trace for lung, with active region shaded. ŽC. HPLC trace for brain, with active region shaded.

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Fig. 5. The HPLC elution profiles for known CDS compounds, in comparison to eluted CDS activity Ž21 min.: ŽA. agmatine Ž6.85 min.; ŽB. noradrenaline Ž7.51 min.; ŽC. histamine Ž5.14 min. and ŽD. adrenaline Ž14.91 min.. GTP, GDP and GMP also eluted with different retention times from lung and brain CDS activity Ždata not shown..

isolation and bioassay of this peak indicated that the compound was responsible for approximately 50% of the CDS activity observed in the initial extracts ŽFig. 4.. Therefore, the same CDS active compound appears to be present in both bovine lung and brain, is approximately three times more abundant in lung than in brain, and is a major contributor to the CDS activity observed in both tissues.

bovine lung or brain. To eliminate the possibility that one of these compounds was responsible for the observed CDS activity we compared the HPLC retention times of these compounds with that observed for the eluted CDS activity. Noradrenaline, adrenaline, histamine and agmatine all eluted with different retention times from the observed CDS activity, indicating that none of these compounds was responsible for the majority of the CDS activity in either bovine lung or brain ŽFig. 5..

3.4. Elimination of noradrenaline, adrenaline, histamine and agmatine as responsible for the obserÕed CDS actiÕity

3.5. Spectroscopic data for CDS

Noradrenaline, adrenaline, histamine and agmatine are known CDSs that could contribute to binding activity in

Isolation from bovine lung Ž2.2 kg wet weight. gave approximately 50 mg of CDS material that appeared pure

Fig. 6. An HPLC trace and UV-spectrum for the new CDS under isocratic conditions. The HPLC trace shows one major peak with a UV-maxima at 280 nm.

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To confirm that the new CDS was not guanosine, GMP, GDP or GTP, we purchased authentic samples of these compounds and obtained their HPLC retention times, MS, NMR spectra, and clonidine displacement binding data. None of the four compounds coeluted with the new CDS or had equivalent spectral data. In addition, the most potent of these compounds ŽGDP. was greater than 100 fold less active than the new CDS. We cannot determine if the CDS compound reported here is the same as the original compound partially isolated by Atlas and Burnstein Ž1984.. Although the UVspectrum of both compounds are similar, the mass spectral data and retention characteristics are different. Mass spectral data can be misleading because compounds do not ionise equally. That is, a minor impurity can preferentially ionise giving the largest mass spectral signal. Also, HPLC retention characteristics can change with pH, especially for a molecule that is charged at one pH and uncharged at another. Therefore, further spectral data is needed to determine if the CDS reported here is the same or different from that reported by the Atlas group ŽAtlas and Burnstein, 1984.. 3.6. Binding data for the new CDS

3

Fig. 7. ŽA. The concentration-dependent displacement of w Hxclonidine by a 2-adrenoceptor binding drugs: ŽI. yohimbine; ŽB. noradrenaline; and Ž`. clonidine from rat cerebral cortical membranes. Error bars are omitted for clarity, experiments were done in triplicate with standard deviation less than 15%. ŽB. The concentration-dependent displacement of w3 Hxclonidine from rat cerebral cortical membranes by CDS extracted from bovine tissue.

by HPLC ŽFig. 6.. However, electrospray mass spectrometry showed two parent ions, at 275 and 489 Da. The presence of two parents is probably due to a minor impurity preferentially ionising in the mass spectrometer. MSMS for the 275 parent showed daughter ions at 191 and 107 Da, while the 489 parent showed daughter ions at 321 and 237, both corresponding to consecutive losses of 84 Da. Both compounds are therefore structurally related, although we cannot determine which is the major component responsible for CDS activity. High resolution mass spectrometry is needed to derive the molecular formula for both components. 1 H-NMR in d 6-DMSO showed one major component, with some aliphatic impurities being present. A singlet at 8.18 ppm and a UV-maxima at 280.6 nm are consistent with the presence of a heteroaromatic moiety, and CH singlets at 5.82, 4.03, 3.98 and 3.92 ppm, a methylene doublet of doublets at 3.64 ppm and three exchangeable hydroxy protons at 5.48, 5.15 and 5.10 ppm strongly indicate the presence of a ribofuranose moiety. Further NMR data is needed for full structure determination of this new CDS.

The bioassay directed isolation was monitored using an a 2-adrenoceptor radioligand binding assay, using displacement of w3 Hx clonidine from rat cerebral cortical membranes. Naphazoline was used to calculate non-specific binding. Clonidine, noradrenaline and yohimbine were used as positive controls ŽFig. 7A.. Partly purified CDS displayed a concentration dependent response at the receptor, consistent with specific activity ŽFig. 7B.. A dose response curve was not obtained for purified CDS because of limited sample availability.

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