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Journal of Lipid Research, Vol. 45, 486-495, March 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from Petromyzon marinus (lamprey) larval liver

K. V. Venkatachalam^1,*, Domingo E. Llanos^*, Kristophe J. Karami^* and Vladimir A. Malinovskii

^* Department of Biochemistry, College of Medical Sciences, Health Profession Division, Nova Southeastern University, Ft. Lauderdale, FL 33328-2018
University of Miami School of Medicine, Department of Biochemistry and Molecular Biology, Miami, FL 33101

Published, JLR Papers in Press, December 1, 2003. DOI 10.1194/jlr.M300346-JLR200

¹ To whom correspondence should be addressed. e-mail: venk{at}nova.edu

	ABSTRACT

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We have isolated, partially purified, and characterized the 5–petromyzonol (5-PZ), (5-cholan- 3, 7, 12, 24-tetrahydroxy-) sulfotransferase (PZ-SULT) from larval lamprey liver. Crude liver extracts exhibited a PZ-SULT activity of 0.9120 pmol/min/mg in juvenile and 12.62 pmol/min/mg in larvae. Using crude larval liver extracts and various 5 ß-cholan substrates and allocholic acid there was negligible activity, however, with 5-PZ and 3-keto-5-PZ the SULT activity was 231.5 pmol/min/mg and 180.8 pmol/min/mg respectively. This established that the sulfotransferase of lamprey larval liver extracts prefers (5 ) substrates and it is selective for hydroxyl at C-24. PZ-SULT was purified through various chromatography procedures. Partially purified PZ-SULT exhibited a pH optimum of 8.0, a temperature optimum of 22°C, and activity was linear for 1h. PZ-SULT exhibited a K_m of 2.5 µM for PAPS and a K_m of 8 µM for PZ. The affinity purified peak PZ-SULT exhibited a specific activity of 2,038 pmol/min/mg. The peak protein upon SDS-PAGE, correlated to an Mw 47 kDa. Photoaffinity labeling with PAP³⁵S, specifically crosslinked the 47 kDa protein, further confirming the identity of PZ-SULT.

Partial amino acid sequencing of the putative 47 kDa PZ-SULT protein yielded a peptide sequence (M)SISQAVDAAFXEI, which possessed an overall (35–40%) homology with mammalian SULT2B1a.

Abbreviations: 3-keto-PZ, 3-keto-petromyzonol; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PZ, petromyzonol; PZS, petromyzonol sulfate; PZ-SULT, petromyzonol sulfotransferase; SULT, sulfotransferase

Supplementary key words petromyzonol sulfate • chemoattractant • pheromone

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammals, bile acids and salts are synthesized in the liver and stored in the gall bladder (1) and aid in solubilizing fats, which facilitates lipolysis (2). A substituent in cyclopentanoperhydrophenanthrene nucleus (3) that is above the plane is termed ß, whereas a substituent that is below the plane is oriented. The hydrogen attached to carbon-5 (C-5) can be either or ß oriented (4). The hydrogen at C-5 results in trans fusion of the ring structure, yielding nearly a planar structure (5, 6), e.g., allocholic acid (ACA)(7). Higher (C-27) bile acids and bile alcohols (also called cholestanes) are found in many organisms. For example, in the small skate Raja erinacea, the major sulfated bile alcohol is scymnol sulfate [3,7,12, 24,26,27-hexahydroxy-5ß-cholestane-26 (27) sulfate] (8). The partial purification and characterization of the enzyme that sulfonates 5ß-scymnol from the liver of the shark Heterodontus portusjackson has been reported (9). In the coelacanth Latimera chalumnae, a 26-sulfate of latimerol (5-cholestane-3ß,7,12,26,27-pentol) and sulfate esters of 5-cyprinol (5-cholestane-3,7,12,26,27-pentol) and 5-bufol (5-cholestane-3,7,12,25,26-pentol) have been reported (10). The identification of cyprinol sulfate from grass carp bile and its toxic effects in rats have been reported (11). The West Indian manatee Trichechus manatus latirostris produces a sulfo-conjugate of 5-cholestane-3,6ß,7-25,26-pentol (12). The presence of various C-27 bile alcohols/salts in fish, amphibians, and mammals has been reported (13, 14).

Cholanes are 24-carbon (C-24) compounds, are similar to cholestanes (C-27), and can also possess hydrogen with an or ß orientation at position number 5, with the usual hydroxyls at 3, 7, and 12 (either or ß) and a carboxyl (bile acid) or hydroxyl (bile alcohol) group at position 24. 5-PZ is a 5-cholan-3,7,12,24-tetrol (7). Petromyzonol (PZ) has been shown to be produced in copious amounts in Petromyzon marinus (lamprey), a jawless, boneless, primitive fish that belongs to the class of Agnatha. The 24-sulfonated derivative of PZ commonly called petromyzonol sulfate, (PZS) (15, 16) and its derivative 5-cholane-(7,12, dihydroxy)-3-one, 24-sulfate (3-keto-petromyzonol-sulfate, 3-keto-PZS, a more potent chemoattractant) have been shown to play a crucial roles as pheromones during the reproductive cycle of lampreys (17). The adult lamprey has been shown to rerurn to the same breeding ground for spawning by smelling the sulfonated derivatives of PZ produced by the larval lamprey. The sulfonate group at the C-24 is very crucial for its bioactivity as a chemoattractant (15–17). The Great Lakes of North America are overpopulated with the vicious lamprey, which, as an adult, feeds on economically important teleosts (salmon, trout, etc.) by sucking the blood from these organisms. Thus, the predator lamprey is a menace to the fishing industry. One of the mechanisms for controlling the overpopulation of this organism is to use 5-PZS and its derivative as the bait to trap adults. Knowledge of the biosynthesis and regulation of the 5-PZS is very crucial to understanding the reproductive physiology of the lamprey so that eventually strategies can be sought to control the lamprey population. This paper is the first to report the isolation, partial purification, and characterization of a novel cholan-specific sulfotransferase (SULT) that is stereo selective (5-cholan) and a regio-selective, C-24 hydroxyl-preferring enzyme from larval livers of lamprey.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
The radiochemical [³⁵S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) for enzyme assays was purchased from NEN Life Science Products or ARC, Inc. Cholan substrates ACA, 3-keto-PZ, PZS, nordesoxycholic acid (NDC), and 5-PZ were purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). 5-PZ was also purchased from Cayman Co. (Ann Arbor, MI). 5ß-24-ol, 5ß-PZ was purchased from Steraloids, Inc. (Newport, RI). Cholic acid (CA), lithocholic acid (LCA), cholesterol, and deoxylithocholic acid (DLCA) were purchased from Sigma-Aldrich, Inc. DEAE ion exchange matrix (Macro-prep DEAE support) was purchased from Bio-Rad (Hercules, CA). Thin-layer chromatography PE silica gel G plates were obtained from Whatman (Clifton, NJ). Nu PAGE, 12% Bis-tris gels, and SDS-PAGE prestained protein molecular weight standards were obtained from Invitrogen (Carlsbad, CA). Larval and juvenile lamprey livers were shipped in dry ice periodically by Hammond Bay Biological Station, Millersburg, MI. ATP, 3'-phosphoadenosine 5'-phosphate (PAP) column affinity matrix, Sephadex (G-100-50) (exclusion limit >100,000 molecular mass) for gel filtration, and all other common biochemicals were also purchased from Sigma-Aldrich, Inc.

Methods
PZ-SULT assay The assay was performed in a total volume of 10 µl. The assay consisted of 3 µl of reaction buffer [150 mM Tris-HCl (pH 8.0), 50 mM KCl, 15 mM MgCl₂, 3 mM EDTA, 45 mM dithiothreitol (DTT)], 4 µl enzyme preparation, 1 µl 50 mM ATP, 1 µl PAP³⁵S (0.16 µCi/0.09 nMol), and 1 µl of 7.68 mM PZ. The typical reaction was carried out at 22°C for 5 min and stopped by placing reaction tubes in boiling water for 5 min. The contents were briefly centrifuged, and 1 µl aliquots were transferred to silica gel thin-layer chromatography plates and chromatographed using chloroform-methanol-water (70:26:4; v/v/v) as the solvent system. Following chromatography, the thin-layer chromatography plates were dried and exposed overnight to X-ray film (Eastman Kodak Co.). The respective PZS spots were cut out and the radioactivity determined by liquid scintillation.

Isolation of PZ-SULT All operations were carried out at 4°C–15°C. Frozen larval livers (1.39 g; 7–8 mg/larval liver) were ground in 3 ml of homogenization buffer [100 mM Tris-HCl (pH 8.0), 1 mM DTT, and 1 mM EDTA, protease inhibitor cocktail III from Calbiochem (San Diego, CA)] with a pinch of sand using a mortar and pestle. The mortar was rinsed with 2 ml of homogenization buffer, and the liquid contents were pooled with the rest of the homogenates. Centrifugation was performed in a Beckman ultracentrifuge (model L7-65) using a Ti-70.1 rotor. The homogenate was first centrifuged at 100 g. The resulting supernatant was then subjected to centrifugation at 10,000 g. The supernatant was then subjected to 100,000 g, and the resulting soluble supernatant was used for PZ-SULT purification.

PZ-SULT purification The soluble extracts were applied onto a 5 ml ion exchange matrix (Macro-prep, DEAE support) and then washed with buffer A [20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM DTT]. The column was eluted at a rate of 1 ml/min with a step gradient of buffer A containing increasing concentrations of NaCl ranging from 0.05 M to 0.4 M. Approximately 10 ml of buffer eluate per step gradient was collected. Protein contents in fractions were measured using the dye binding method described by Bio-Rad. Four microliters of each fraction was assayed for PZ-SULT activity, as described previously. PZ-SULT activity fractions (30–35) (total of 6 ml) were pooled and applied to a Sephadex G-100 gel filtration column (80 ml bed volume). The column was eluted with buffer A, and 1 ml fractions were collected at a rate of 12 min per fraction. PZ-SULT activity fractions (33–47) were further pooled and concentrated using an Ultrafree-4 centrifugal filter unit [molecular mass cutoff 5 kDa, (Millipore)] to 1.8 ml. A portion of the fraction (1.6 ml) was applied onto a 2 ml affinity column matrix, PAP immobilized on cross-linked 4% beaded agarose (Sigma-Aldrich). The enzyme preparation was passed through at least five times, and after the final pass, the proteins were allowed to bind to the matrix for >60 min. The column was then eluted with buffer A containing a salt step gradient, 5 ml each of 0.05 M to 0.4 M NaCl. PZ-SULT-active fractions from the PAP column were resolved by SDS-PAGE using 12% Bis-tris gels. Gels were stained with Silver-Express according to instructions provided by Invitrogen.

Photoaffinity labeling Larval liver 100,000 g supernatant was purified through DEAE ion exchange chromatography, in a procedure similar to those described earlier. For subsequent PAP affinity column purification, only the very peak fraction was included, and the rest of the peak was eliminated. This avoided carry-over of many contaminating proteins. The very peak fraction from the PAP affinity column-purified enzyme fraction and a nonpeak fraction were concentrated using an Ultrafree-4 centrifugal filter unit [molecular mass cutoff 5 kDa (Millipore)]. A peak PZ-SULT-active fraction and a nonpeak PZ-SULT-inactive fraction were used for photoaffinity labeling. The reaction was performed in a total volume of 50 µl. The reaction consisted of 45 µl affinity-purified proteins in buffer containing 20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM DTT, 0.20 M NaCl, and 5 µl of PAP³⁵S [6.76 µCi (specific activity of 3.00 Ci/mmol)]. In a cold chase experiment, the reaction contained an additional 0.36 mM nonradioactive PAPS. Photoaffinity labeling was performed using UltraVette UV cuvettes from BrandTech Scientific, Inc., (Essex, CT). The samples were irradiated for 10 min (672,000 µJ/cm²) at 25°C using CL-1000 UV cross-linker from UVP, Inc., (Upland, CA). The reaction was chilled on ice and stopped by adding 6 µl of SDS-PAGE loading buffer from Invitrogen. The proteins were then denatured by heating the samples at 95°C for 5 min. A 35 µl aliquot was loaded onto 12% Bis-tris gels. Gels were stained with Silver-Express according to instructions provided by Invitrogen. For autoradiography, the gels were dried and exposed to X-ray film (Xomat AR, Eastman Kodak Co.) for 6 days.

Protein sequencing The proteins from SDS-PAGE were electroblotted onto Immobilon-P membranes and stained with Ponceau S. A 47 kDa band was cut and amino terminally sequenced by the Edman degradation method using a Procise 491 protein sequencer (Applied Biosystems, Inc.). No sequence was obtained, perhaps due to NH₂-terminal blocking. The membranes were cut into small pieces and wetted with methanol, then treated with 100 µl of 2% cyanogen bromide in 70% formic acid under nitrogen for 24 h in the dark. The membranes were then extracted four times with 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) for 12 h and two times with 20% acetonitrile without TFA. The supernatants containing the digested peptides were removed. The membrane remaining after acetonitrile-water-TFA extraction was used for sequencing.

Homology analysis The 14-amino acid PZ-SULT peptide was aligned with SULT using DNA and protein sequence analysis software OMIGA 2.0 (Oxford Molecular Co., Madison, WI). GenBank-deposited mammalian SULTs used in the analysis were SULT2B1a (protein ID, AAC78553.1), SULT2B1b (protein ID, AAC78554.1), SULT2B1a (protein ID, AAC78498.1), and SULT2A1 (protein ID, NP 003158.2).

	RESULTS

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Isolation of PZ-SULT
PZ-SULT activity in juvenile versus larval liver Liver tissue extracts (100 g supernatant) from juveniles and larvae were tested for PZ-SULT activity. Tissue extracts from both juveniles and larvae, without the exogenously added 5-PZ cosubstrate, exhibited negligible activity. There was 10-fold higher PZ-SULT activity with 5-PZ in larvae (12.62 pmol/min/mg) compared with juvenile activity (0.9120 pmol/min/mg) (Fig. 1) . Using enzymatically concentrated preparations (based on PZ-SULT activity) of larval liver tissue extracts, various analogs were tested for SULT activity. The analogs tested included cholesterol, which is the precursor of all C-27 cholestane, 5-cholan substrates (5-PZ, 5-PZS, 3-keto-5-PZ, and ACA), and 5ß-cholan substrates [5ß-PZ, 5ß-cholan-24ol (5ß-24ol), NDC, CA, DLCA, and LCA]. Neither the common 5ß bile acid compounds (CA, NDC, DLCA, and LCA) nor 5ß-24-ol, a bile alcohol, formed sulfonated products. 5ß-PZ possessed a negligible activity of 25.3 pmol/min/mg. With 5-PZ, the extract contained a SULT activity of 231.5 pmol/min/mg. With 3-keto-5-PZ, the activity was 180.8 pmol/min/mg. All other substrates, including ACA, exhibited only background activity for sulfonation (Fig. 2) . ACA is the same as the 5-PZ, except that at position 24, it has a carboxyl group instead of a hydroxyl group. The PZ-SULT activity exhibits a temperature optimum of 22°C (Fig. 3A) . The activity was stable and linear for 1 h of incubation at 22°C at pH 8.0 (Fig. 3B) and a pH optimum of 8.0 (Fig. 3C). The partially purified enzyme, when tested with analogs ACA, 5ß-PZ, and 5-PZS, showed negligible activity, similar to that shown by crude extracts (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Petromyzonol sulfotransferase (PZ-SULT) activity in juvenile and larval liver extracts. Data represent the mean values (n = 3), and error bars indicate ±SD.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Sulfotransferase (SULT) activity in crude larval liver extracts against various compounds. The compounds were dissolved in ethanol. The final concentrations of the substrates in 10 µl enzymatic reactions were: 0.768 mM, 5-PZ; 1.4 mM, petromyzonol sulfate (PZS); 0.14 mM, PZ-24-SO4; 0.423 mM, 5ß-PZ; 0.58 mM, 5ß-cholan-24-ol; 1.02 mM, 3-keto-PZ (3-keto-petromyzonol); 1.22 mM, allocholic acid (ACA); 1.32 mM, nordesoxycholic acid (NDC); 0.784 mM, cholesterol (Cholest); 0.742 mM, CA (cholic acid); 0.805 mM, lithocholic acid (LCA); and 0.772 mM, deoxylithocholic acid (DLCA). Plain ethanol was used in the no substrate (No subs) reaction. Data represent the mean values (n = 2).

View larger version (26K): [in this window] [in a new window]   Fig. 3. A: Effect of temperature on PZ-SULT activity. Reaction at each temperature was performed for 15 min at pH 8.0 according to the standard assay procedures described in Materials and Methods. B: Effect of time on PZ-SULT activity. Reactions at various time periods were performed at 22°C, pH 8.0. The reactions were then stopped by heat inactivation by boiling at 95°C, and the contents were processed according to the standard assay procedures described in Materials and Methods. C: Effect of pH on the PZ-SULT activity. Reactions were performed for 15 min at 22°C, using reaction buffers of various pHs that contained, respectively, ingredients used in the standard assays described in Materials and Methods. [The following buffers were used for various pHs: citrate reaction buffer (pH 4.0, 5.0, and 6.0); phosphate buffer (pH 6.0 and 7.0); Tris-HCl (pH 8.0 and 9.0), and glycine (pH 9.0, 10.0, and 11.0).]

View larger version (13K): [in this window] [in a new window]   Fig. 4. DEAE–macro prep elution profile of PZ-SULT. Crude larval liver 100,000 g supernatant was purified through DEAE ion exchange columns. The columns were washed with buffer A with no salt to remove unbound proteins and then eluted with buffer A containing NaCl (0.05 M to 0.4 M) consisting of 10 ml step gradients. Approximately 1 ml fractions were collected, and a 4 µl aliquot was assayed for PZ-SULT activity. Protein amounts were determined by Bio-Rad dye binding assay. The protein contents per µl are represented (as described in Materials and Methods). Peak PZ-SULT activity eluted at 0.3 M NaCl concentration.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Sephadex G-100 gel filtration chromatography purification of PZ-SULT. DEAE fractions 30–35 that contained PZ-SULT activity were pooled and loaded onto the gel filtration column. The column was eluted with buffer A containing 0.05 M NaCl at a rate of 1 ml/12 min. PZ-SULT activity in a 4 µl aliquot from 1 ml fraction was measured. Protein per 1 µl is represented.

View larger version (12K): [in this window] [in a new window]   Fig. 6. 3'-Phosphoadenosine 5'-phosphate (PAP) affinity column chromatography purification of PZ-SULT. PZ-SULT fractions (33–47) from gel filtration chromatography were pooled and concentrated using an Amicon ultra filtration unit (molecular mass cutoff 5 kDa), and a 1.6 ml concentrate was passed through the PAP column five times. The final pass through was allowed to bind for >60 min. The column was washed and eluted with 5 ml each of buffer A with NaCl (0.05 M to 0.4 M) step gradient. The activity that did not bind to the column eluted in buffer containing a low-salt wash of 0.05 M NaCl. The bound PZ-SULT activity eluted in buffer containing 0.2 M NaCl.

View larger version (42K): [in this window] [in a new window]   Fig. 7. SDS-PAGE analysis of PAP affinity column-purified PZ-SULT. Fractions 13 and 14, which contained PZ-SULT activity, and fraction 15, which contained no PZ-SULT activity, were analyzed by 12% Bis-tris SDS-PAGE gels and stained with silver. Prestained molecular mass marker from Invitrogen, followed by lanes 1 and 2 corresponding to PZ-SULT-active fractions containing the major protein (47 kDa), indicated by the arrow. Lane 3, which contained no PZ-SULT activity, lacks the 47 kDa band.

View larger version (143K): [in this window] [in a new window]   Fig. 8. Photoaffinity labeling of peak PZ-SULT-activity protein. A: SDS-PAGE analysis for purity of the PZ-SULT preparation used for photoaffinity labeling: 1, molecular mass marker; 2, empty lane; 3, PZ-SULT peak fraction (0.67 µg); 4, fraction containing no PZ-SULT activity (0.25 µg). B: Autoradiograph of the photoaffinity labeling: 1, empty lane; 2, peak PZ-SULT fraction showing the cross-linked, labeled 47 kDa protein; 3, corresponds to the fraction containing no PZ-SULT activity.

View larger version (25K): [in this window] [in a new window]   Fig. 9. A: PZ-SULT kinetic analysis against 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as substrate. DEAE chromatography followed by PAP affinity column. Purified enzyme was assayed for PZ-SULT against various concentrations of PAPS, as described in Materials and Methods. Data points represent the average of two independent experiments. Double-reciprocal transformations are shown in the insets. B: PZ-SULT kinetic analysis against petromyzonol (PZ) as substrate. DEAE chromatography followed by PAP affinity column. Partially purified enzyme was assayed for PZ-SULT against various concentration of PZ, as described in Materials and Methods. Data points represent the average of two independent experiments. Double-reciprocal transformations are shown in the insets.

View larger version (39K): [in this window] [in a new window]   Fig. 10. Amino acid homology of lamprey PZ-SULT with mammalian SULT. PZ-SULT, SULT2B1a (aa residues 31–45; protein ID, AAC78553.1 or AAC78498.1), SULT2B1b (aa residues 46–60; protein ID, AAC78554.1), and SULT2A1 (aa residues 20–34; protein ID, NP003158.2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

When most active larval liver extracts were tested with various analogs for sulfonation, none of the 5ß substrates formed significant sulfonated products, except for 5ß-PZ, which had slightly above background (25.3 pmol/min/mg), which is about ten times less than 5-PZ (231.5 pmol/min/mg). This clearly established that the larval liver extracts contained a stereo-specific (5)-preferring enzyme compared with 5ß substrates. Among the 5 substrates tested, only 5-petromyzonol and 3-keto-5-PZ formed significant sulfonated products. 3-Keto-5-PZ is the same as 5-PZ, except that the 3-keto-PZ contains a keto group at the third position. This clearly indicates that the substitution at position 3 does not affect the sulfonating activity of SULT and that the sulfonation is perhaps occurring at the other hydroxyl positions. The exact biosynthetic sequence of the 5-PZS and of the more potent chemoattractant 3-keto-5-PZ have not been elucidated. The pathway for the biosynthesis of C-27 5-cyprinol has been proposed, and some of the crucial dehydrogenases have been identified (18). On the basis of the SULT activity levels of the two substrates, 5-PZ (231.5 pmol/min/mg) and 3-keto-PZ (180.8 pmol/min/mg), one can speculate that 5-PZS is formed first and then the 3-dehydrogenase oxidizes the 5-PZS into 3-keto-5-PZS, the potent chemoattractant. ACA is the same as the 5-PZ, except that position 24 is carboxyl instead of a hydroxyl group. ACA did not form any sulfonated products, which clearly established that the PZ-SULT prefers the hydroxyl group at the C-24 position for sulfonation. In addition, 5-PZ-24SO₄, which has three free unconjugated hydroxyl groups at positions 3, 7, and 12 and has the potential to be sulfonated, did not form any radioactive sulfonated products. Thus, we conclude that the PZ-SULT present in larval liver extract is a stereo-specific (5-PZ-preferring) and also regio-selective (C-24 hydroxyl-preferring) enzyme for sulfonation.

The affinity column-purified PZ-SULT fraction exhibited a specific activity of 2,038 pmol/min/mg and correlated with a band of 47 kDa analyzed by SDS-PAGE. This is the first report on the isolation, purification, and characterization of a novel 5-cholane (C-24)-specific SULT from fish. The only other known partially purified and characterized SULT from fish is a cholestane (C-27) type, 5ß-scymnol SULT, from shark (Heterodontus portusjacksoni). The 5ß-scymnol SULT enzyme exhibits a molecular mass of 40–45 kDa (9).

The molecular mass is very close to that of the 5-cholane type, petromyzonol SULT, although evolutionarily, lampreys (boneless) are far removed from sharks (bony fish). Various mammalian SULTs range in molecular mass from 30 to 45 kDa (19).

When the 14-amino acid peptide sequence (M)SISQAVDAAFXEI corresponding to putative PZ-SULT was compared with mammalian SULT2A1, SULT2B1a, and SULT2B1b using Omiga DNA and a protein software analysis program, the analysis yielded a remarkable overall homology of 35–40%. It is particularly interesting that the amino acid residue SISxA of the PZ-SULT is highly conserved in mammalian SULT2B1a. The cytosolic SULT superfamily is divided into five families, one of which (SULT2) is primarily engaged in the sulfoconjugation of neutral steroids and sterols. The mammalian hydroxysteroid SULTs have sizes that range from 282 to 295 amino acids, whereas SULT2B1a and SULT2B1b consist of 350 and 365 amino acids, respectively. Overall, the SULT2A1 and SULT2B1 isozymes are 37% identical at the amino acid level (20). Using a purified peak PZ-SULT activity fraction, photoaffinity labeling with PAP³⁵S was performed to confirm the identity of PZ-SULT. Photoaffinity labeling cross-linked the 47 kDa polypeptide, further confirming the isolation of the PZ-SULT polypeptide. Similar photoaffinity labeling of human cytosolic and recombinant SULTs using 2-azidoadenosine 3', 5'-[5'-³²P]bisphosphate has been reported. (21) 5-PZ-SULT exhibited a K_m of 8 µM for PZ and 2.5 µM for PAPS.

5ß-Scymnol SULT, in comparison, exhibited a K_m of 4 µM for PAPS and 14 µM for 5ß-scymnol (9). These values are very close, although they are entirely different enzymes from two different fish: one is a cholestane (C-27) SULT and the other is a cholane (C-24) SULT. In this paper, we have described a novel 5-cholane-specific SULT-preferring hydroxyl at the C-24 and have reported the characterization of the biochemical properties. Understanding the overall biosynthesis of 5-PZS is critical to understanding the reproductive cycle of lampreys and to arriving at possible strategies for controlling the overpopulation of lampreys in the Great Lakes.

	ACKNOWLEDGMENTS

 
The authors thank Cody Smith for technical help and Dr. Judy Mack and Dr. Susan Abramson of Cleveland Clinic of Florida for their helpful comments. The authors thank the University of Miami Protein Core Facility for their help in protein sequencing. This study was supported by a grant from the U.S.A. Great Lakes Fisheries Commission.

Manuscript received August 7, 2003 and in revised form November 18, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Borgstrom, B., J. A. Barrowman, and M. Lindstrom. 1985. Roles of bile acids in intestinal lipid digestion and absorption. In Sterols and Bile Acids. Elsevier, Amsterdam. 405–425.

2. Borgström, B. 1992. Influence of bile salt, pH and time on the action of pancreatic lipase: physiological implications. J. Lipid Res. 5: 522–531.

3. Hofmann, A. F., J. Sjovall, G. Kurz, A. Radominska, C. D. Schteingert, G. S. Tint, Z. R. Vlahcevic, and K. D. Setchell. 1992. A proposed nomenclature for bile acids. J. Lipid Res. 33: 599–604.[Abstract]

4. Carey, M. C. 1985. Physical-chemical properties of bile acids and their salts. In Sterols and Bile Acids. Elsevier, Amsterdam. 345–403.

5. Haselwood, G. A. D. 1967. Bile salt evolution. J. Lipid Res. 8: 535–550.[Abstract]

6. Hay, D. W., and M. C. Carey. 1990. Chemical species of lipids in bile. Hepatology. 12 (Suppl.): 6–12.

7. Haselwood, G. A. D., and L. Tokes. 1969. Comparative studies of bile salts. Bile salts of the lamprey Petromyzon marinus L. Biochem. J. 114: 179–184.[Medline]

8. Karlaganis, G., S. E. Bradley, J. L. Boyer, A. K. Batta, G. Salen, B. Egestad, and J. Sjovall. 1989. A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea). J. Lipid Res. 30: 317–322.[Abstract]

9. Macrides, T. A., D. A. Faktor, N. Kakafatis, and G. Amiet. 1994. Enzymic sulfation of bile salts. Partial purification and characterization of an enzyme from the liver of the shark Heterodontus portusjacksoni that catalyzes the sulfation of the shark bile steroid 5ß-scymnol. Comp. Biochem. Physiol. 1078: 461–469.[CrossRef]

10. Kihira, K., Y. Akashi, S. Kuroki, J. Yanagisawa, F. Nakayama, and T. Hoshita. 1984. Bile salts of the coelacanth, Latimera chalumnae. J. Lipid Res. 25: 1330–1336.[Abstract]

11. Hwang, D-F., Y-H. Yeh, Y-H. Lai, and J-F. Deng. 2001. Identification of cyprinol and cyprinol sulfate from grass carp bile and their toxic effects in rats. Toxicon. 39: 411–414.[Medline]

12. Kuroki, S., C. D. Schteingart, L. R. Hagey, B. I. Cohen, E. H. Mosbach, S. S. Rossi, A. F. Hofmann, N. Matoba, M. Une, T. Hoshita, and D. K. Odell. 1988. Bile salts of the West Indian manatee, Trichechus manatus latirostris: novel bile alcohol sulfates and absence of bile acids. J. Lipid Res. 29: 509–522.[Abstract]

13. Hoshita, T. 1996. Comparative biochemical studies of cholanoids. Yakugaku Zasshi. 116: 71–90.[Medline]

14. Une, M., and T. Hoshita. 1994. Natural occurrence and chemical synthesis of bile alcohols, higher bile acids, and short side chain bile acids. Hiroshima J. Med. Sci. 43: 37–67.[Medline]

15. Li, W., P. W. Sorensen, and D. D. Gallaher. 1995. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae. J. Gen. Physiol. 105: 569–587.[Abstract/Free Full Text]

16. Polkinghorne, C., J. M. Olson, D. G. Gallaher, and P. W. Sorensen. 2001. Larval sea lamprey release two unique bile acids to the water at a rate sufficient to produce detectable riverine pheromone plumes. Fish Physiol. Biochem. 24: 15–30.[CrossRef]

17. Li, W., A. P. Scott, M. J. Siefkes, H. Yan, Q. Liu, S-S. Yun, and D. A. Gage. 2002. Bile acid secreted by male sea lamprey that acts as a sex pheromone. Science. 296: 138–141.[Abstract/Free Full Text]

18. Hoshita, T. 1969. Stereo-bile acids and bile alcohols. CV. Conversion of 7alpha,12-alpha-dihydroxycholest-4-en-3-one to 5-alpha-cyprinol by carp liver. J. Biochem.(Tokyo). 66: 313–319.[Abstract/Free Full Text]

19. Strott, C. A. 2002. Sulfonation and molecular action. Endocr. Rev. 23: 703–732.[Abstract/Free Full Text]

20. Strott, C. A., and Y. Higashi. 2003. Cholesterol sulfate in human physiology: What's it all about? J. Lipid Res. 47: 1268–1278.

21. Radominska, A., R. R. Drake, X. Zhu, M. E. Veronese, J. M. Little, S. Nowell, M. E. Mcmanus, R. Lester, and C. N. Falany. 1996. Photoaffinity labeling of human recombinant sulfotransferases with 2-azidoadenosine 3'5'-[5'-³²P]bisphosphate. J. Biol. Chem. 271: 3195–3199.[Abstract/Free Full Text]

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