Liver Of Catfish Research Articles

Effects of anisotonicity on pentose-phosphate pathway, oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish, Clarias batrachus.

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of 14CO2 production from [1-14C]glucose by 110 and 70%, respectively, from the basal level of 18.4 +/- 1.2 nmol/g liver/min from the perfused liver of Clarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of 14CO2 production from [1-14C]glucose by 34%. 14CO2 production from [6-14C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 +/- 0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on 14CO2 production from [1-14C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 mmol/l). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.

SUBSTRATE SPECIFICITY AND INHIBITION STUDIES ON AFRICAN CATFISH (<i>Clarias gariepinus</i>) LIVER GLUTATHIONE S-TRANSFERASE

The effect of some inhibitors of glutathione transferases was examined on a purified glutathione transferase from the liver of the African catfish (Clarias gariepinus). The ability of the enzyme to catalyze the conjugation of glutathione (GSH) with a variety of other compounds was also examined. The glutathione transferase (GST) from the African catfish was inhibited by all the compounds examined. Cibracron blue and triphenyltin chloride were the most potent inhibitors with I50 (concentration of inhibitors causing 50% inhibition) of 0.0875μM and 0.0525μM respectively. The enzyme displayed broad substrate specificity. The enzyme was able to catalyze the conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid , 4-nitrobenzylchloride, p-nitrophenylacetate and hydrogen peroxide. The results obtained from this work suggest that the glutathione transferase from the liver of African catfish might possibly belong to the Pi and Mu class of isoenzymes and that the enzyme is homodimeric. Key Words: Glutathione transferase; African catfish; Clarias gariepinus; substrate specificity; isoenzyme(s) Global Jnl Pure & Applied Science Vol.10(1) 2004: 179-182

Fenvalerate-induced changes in a catfish, Clarias batrachus: metabolic enzymes, RNA and protein

We studied the effects of a sublethal concentration of pyrethroid insecticide fenvalerate on metabolic enzymes, RNA and protein of brain, liver and skeletal muscle of the freshwater catfish, Clarias batrachus. Exposure to fenvalerate gradually decreased the activity of citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogenase (LDH) in brain, liver and skeletal muscle up to 21 days. The maximum decrease in enzyme activity was 23–47%. Withdrawal of fenvalerate from the medium for 21 days restored enzyme activity to their control level in all three tissues. RNA and protein content in brain, liver and skeletal muscle decreased significantly with exposure of fenvalerate up to 21 days. The maximum decrease in RNA and protein was 22–32%. Withdrawal of fenvalerate from the medium for 21 days restored the RNA and protein contents to control levels. The present study suggests that fenvalerate impairs cellular metabolism and its biochemical effects are reversible after withdrawal of fenvalerate.

Persistent organic pollutants and histological lesions in Mayan catfish Ariopsis assimilis from the Bay of Chetumal, Mexico

Livers of catfish ( Ariopsis assimilis) from the Bay of Chetumal were analyzed for organochlorine compounds and hydrocarbons as part of a study to diagnose the environmental health of the Bay after a catfish mass mortality that occurred in 1996. The presence of histological lesions in several organs of the fish as result of chemical exposure was also evaluated. The concentrations of organic pollutants found in the Bay may be considered high if compared to the levels reported for sites affected by chemical pollution. High prevalences of cellular alteration histopathologies were found in liver, including hepatic tumors. The presence of some lesions may be related statistically to environmental pollution in the Bay, specially with chlorinated compounds.

Starvation-induced impairment of metabolism in a freshwater catfish.

Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation-induced macromolecular changes indicate impairment of metabolism in fish.

Microwave‐assisted catfish liver oil extraction and FA analysis

AbstractFA profiles of catfish liver oils were analyzed after microwave‐assisted (without solvent extraction) and/or conventional (with solvent extraction) preparation methods. Microwave heating of the smaples was performed at 100, 80, 60, or 40% power at 1,000 W and 2,450 MHz, each for 80, 60, 40, or 20 s. Significant differences in the content of recovered FA were observed among the microwave‐heated samples, except for C20∶0 and C20∶4. Recovery of C16∶0, C20∶0, and C20∶4 from the samples analyzed by the microwave‐assisted method was lower than that of the samples analyzed by the conventional method. Much greater recovery was observed for C18∶1, C18∶2, and C22∶6; however, the recovery was not different from or was only slightly lower than that of the conventional method when microwave heating was set at 40% power for 20 s. This was also observed for the total unsaturated or saturated FA. Compared to other microwave treatments, heating at 100% power for 80 s yielded the greatest recovery of C14∶0, C18∶0, C18∶1, C18∶2, C18∶3, C20∶1, C20∶2, and C22∶6.

Cloning and sequencing of cytochrome P450 2X1 from channel catfish (Ictalurus punctatus)

Previous purification and immunochemical studies in livers of channel catfish indicated the presence of at least four cytochrome P450 (CYP) 2-like isoforms. Sequencing of the first 18 amino acids of one purified form indicated a CYP2 isoform. From this N-terminal sequence and other published CYP2 sequences from fish, primers were designed and a full-length CYP cDNA was identified from reverse-transcribed catfish liver mRNA. 5′ and 3′ RACE was used to obtain an open reading frame of 1470 bp encoding a 490 amino acid protein (approximately 57 kD). CYP2X1 was most identical to Fundulus heteroclitus CYP2P2 (41%); CYP2N2 (40%): and CYP2N1 (39%).

MERCURY ACCUMULATION IN CATFISH (ICTALURUS FURCATUS AND I. PUNCTATUS) FROM THE SOUTHWESTERN TENNESSEE RIVER VALLEY

Mercury from an industrial source was introduced in the 1960's into Pickwick Lake, an impoundment on the Tennessee River in northwest Alabama. Liver and muscle tissue of catfish (Ictalurus furcatus) collected from this lake were analyzed to determine if Hg continues to be a contaminant in this system. Low concentrations of mercury were detected in all of these fish. Catfish (Ictalurus furcatus) from an adjacent upstream lake and catfish (Ictalurus punctatus) from five surrounding farm ponds were also collected. Tissue levels of Hg were found to be higher in fish from the upstream lake and from the farm ponds than those from Pickwick Lake. Although Hg is still accumulating in fish in Pickwick Lake, the source does not appear to be from the historic industrial releases. A possible source for the Hg at all the sites is atmospheric deposition from local coal-burning electrical generation plants. Mercury residues in muscle tissue did not exceed the USFDA limit for any of the catfish collected in this area.

Identification and Properties of a Gs Protein in Catfish Liver Membranes

The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5′-O-(γ-thiotriphosphate) (GTPγS) (maximal activation of about eightfold at 10−5 M; half-maximal activation at 1.31 × 10−7 M), and reduced by the competitive inhibitor of GTP, GDPβS (70% maximal inhibition at 10−4 M; half-maximal inhibition at 1.98 × 10−7 M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 μM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [35S]GTPγS binding assay. At 100 μM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20°, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPγS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 μM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a Gsα-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-Gsα antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein αs subunits in the liver of an ectothermal vertebrate.

P-glycoprotein in the catfish intestine: inducibility by xenobiotics and functional properties

The p-glycoprotein (pgp)-mediated multixenobiotic resistance (MXR) mechanism of aquatic animals has been associated with protection against pollution. Recent studies in mammals suggest that intestinal pgp may modulate intestinal bioavailability of dietary xenobiotics. In order to further delineate this mechanism in the catfish, these studies: (1) examined the pgp-related distribution in the intestine and liver of catfish, (2) evaluated the MXR response following exposure to various dietary xenobiotics and a prototypic pgp inducer and (3) evaluated pgp functional activity in membrane vesicles, using prototypic substrates and inhibitors. For this purpose, catfish were exposed in vivo to the pgp inducer vincristine (VIN), and the xenobiotics β-naphthoflavone (BNF), benzo[ a]pyrene (BaP), and 3,4,3′,4′-tetrachlorobiphenyl (TCB). Membrane vesicles, prepared from liver and intestine (proximal and distal sections) of control and exposed catfish, were subjected to SDS PAGE, Western Blot, and detection with the pgp C219 monoclonal antibody. Transport activity was evaluated in vitro using the pgp substrate [ 3H]vinblastine (VBL), and the pgp inhibitor verapamil (VP). Immunoblot studies demonstrated a pgp-related protein of approximately 170 kDa in the intestine and liver of catfish. This protein appears to be very susceptible to degradation, and was present in higher levels in the liver, in comparison to the intestine, where regional differences were not observed. Dietary exposure to the pgp substrate VIN, or the xenobiotics BNF, BaP, and TCB, did not appear to affect pgp-related reactivity. Transport studies with VBL indicate that the pgp-related protein of the catfish intestine displays classic pgp-mediated multidrug resistance (MDR) characteristics, such as energy-dependency, and sensitivity to VP. These studies suggest that the pgp-related protein in the catfish intestine and liver is not only immunochemically, but also functionally related to the mammalian MDR. Moreover, the results presented indicate that pgp-related reactivity and transport in intestinal vesicles of catfish may be influenced by factors including method sensitivity, sample collection, sample preparation, and immunoblot conditions.

THE PROTECTIVE EFFECT OF ASCORBIC ACID ON THE HISTOLOGICAL CHANGES CAUSED BY THE HERBICIDE (RONSTAR) IN NILOTIC CATFISH : CLARIAS LAZERA ( C GARIEPINUS)

Ronstar was applied at concentration s of 2.0,5.75 and 9.5 mg/L. Histological alterations were determined in liver and kidney of the catfish Clarias lazera. The histological changes in liver of catfish C lazera intoxicated with different doses of Ronstar, included dilation of the blood veins and sinusoids, vacuolation in the cytoplasm and pyknosis in the nuclei of the hepatocytes, haemorrhage is also noticed in the veins and sinusoids. The phagocytic Kupffer and endothelial cells are also abundant as an immunoresponse reaction. The vacuolation is the most frequent histological alteration observed at all tested doses of Ronstar. Ascorbic acid (lOmg/lkg of body *weight)was reported to reduce some of the histological damage occurred in the liver tissue. It alleviated the rate ofpyknosisand vacuolationThe rate of ascorbic acid healing effect depends on the Ronstar tested dose. Ronstar induced different histological alterations in the kidney of the catfish C lazera The glomeruli became expanded and their capillaries were extended, the subcapsular space enlarged or disappeared. The haemopoietic tissue exhibited some vaculation and the renal tubules were swollen or degenerated. Some of them took an irregular shape .The expanded glomeruli with extended capillaries

Confirmation of catfish, Ictalurus punctatus (Rafinesque), mortality from Microcystis toxins

Unexplained deaths of pond‐grown catfish have occurred for many years. At least some of these mortalities could be from cyanobacteria toxins ingested during feeding on floating diets or passively assimilated through gills during breathing. Recently we were able to document algal production and subsequent ingestion of these toxicants by catfish during a mortality event. The causative organism, Microcystis aeruginosa, was the dominant species within the phytoplankton community during the cooler autumn‐winter season. Pond conditions included a drop in water temperature by c. 5 °C during the 10 days preceding the fish mortalities. Microcystin‐LR, a hepatotoxin produced by Microcystis, was detected in water samples and in catfish liver tissue. Fish exposed to pond water containing this toxic bloom were killed within 24 h. Necropsy of fish revealed congested liver and spleen tissues. The combination of clinical signs, detection of microcystin LR in water and in liver, and death of fish exposed to pond water supports the diagnosis of microcystin toxicosis. More research is needed to identify specific environmental conditions initiating toxin production to model and predict occurrence of these toxic algal blooms.

Novel Transcripts of the Estrogen Receptor α Gene in Channel Catfish

Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERα cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERα variants of different sizes. Relative to the catfish ERα (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERα (36 residues longer) and Short-ERα (389 residues shorter). The 5′-end of Long-ERα cDNA is identical to that of Medium-ERα but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERα binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ERα but lower than reported for catfish ERβ. Short-ERα cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERα RNAs that include the size range of the ERα cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERα cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERα antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERα antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERα mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may encode functional ERα or related proteins that modulate ERα or ERβ activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may be to participate in the regulation of ER gene expression.

CONFIRMATION OF CATFISH MORTALITIES RESULTING FROM MICROCYSTIN PRODUCED DURING MICROCYSTIS BLOOMS

Enigmatic deaths of finfish have occurred in freshwater systems for many years. Some of these fish mortalities can be attributed to the accumulation of cyanobacterial toxins during ingestion of floating feeds or passively through opercular ventilation. We recently were able to document the accumulation of microcystin and resulting catastrophic mortality of pond‐raised catfish (Ictalurus punctatus). The causative organism, Microcystis aeruginosa, was the dominant phytoplankton component within the cooler fall‐winter season. Microcystin, a hepatotoxin produced by Microcystis, was detected in water and catfish liver samples. Fish exposed to pond water containing this bloom were killed within 24 h. Necropsy of fish exposed to pond water revealed congested liver and spleen tissues. Environmental conditions associated with several similar bloom events suggest several predictive options to forecast conditions supporting toxin formation, particularly temperature.

Conservation of a glutathione S-transferase in marine and freshwater fish

We have previously reported the isolation and cloning of glutathione S-transferase (GST) cDNAs from two marine fish, English sole ( Pleuronectes vetulus) and starry flounder ( Platichthys stellatus), that exhibited >95% identity to plaice ( Pleuronectes platessa) GST-A Aquatic Toxicol., 44, 171–182]. In the present study, we have used reverse transcription–polymerase chain reaction (RT–PCR) analysis to isolate a 471 nucleotide GST-like cDNA from largemouth bass ( Micropterus salmoides) liver. Sequence identity of the largemouth bass partial cDNA to plaice GST-A was approximately 90%. Northern blotting analysis using the partial GST cDNA from English sole as a probe detected a single band of approximately 1 kb in English sole liver and a slightly larger GST-like band that was highly expressed in largemouth bass liver. In addition, a faint band of similar size was recognized in brown bullhead ( Ameriurus nebulosus) liver, but not in channel catfish ( Ictalurus punctatus) liver. In conclusion, we have extended our studies of GST expression in flatfish and have isolated an additional GST-A-like cDNA from a largemouth bass. Conservation of a GST-A like cDNA among certain marine flatfish and freshwater species suggests an important function for this gene.

Isolation and characterization of a novel cytochrome P450 (CYP2 family) isoform from channel catfish

Channel catfish ( Ictalurus punctatus) express at least four isoforms of cytochrome P450 (CYP) that are cross-reactive with anti-CYP2K1 (rainbow trout) antibodies. These proteins appear to be the major constitutive CYPs in the liver of I. punctatus and show distinct sex- and age-specific variations in expression. The objective of the present work was to purify a CYP2-related hepatic CYP from I. punctatus. Hepatic microsomes were prepared from channel catfish liver and solubilized with sodium cholate and polidocanol. CYP proteins were precipitated with polyethyleneglycol and applied to an octylamino-Sepharose CL-4b column. CYP-containing fractions were subsequently subjected to chromatography on DEAE-sepharose, HA-ultragel, and CM-cellulose columns. A single heme protein (ca. 47 kDa), which reacted with anti-CYP2K1 antibodies, was resolved. A sequence for the first 15 amino acids was determined. This sequence showed homology with both CYP2M1 and CP2K1 isoforms. Expression of this band is down regulated by exposure to ethanol and fenitrothion. However, no correlation with several CYP-mediated catalytic activities has been detected as yet.

CDNA sequence and tissue-specific expression of a basic liver-type fatty acid binding protein in adult zebrafish ( Danio rerio)

We have determined the nucleotide sequence for a cDNA clone derived from zebrafish ( Danio rerio) that codes for a fatty acid binding protein (FABP). Amino acid sequence similarity of the zebrafish FABP was highest to FABPs isolated from the livers of catfish, chicken, salamander and iguana. The open-reading frame of the zebrafish FABP cDNA codes for a protein of 14.0 kDa with a calculated isoelectric point of 8.8. The zebrafish liver-type FABP (L-FABP) cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases HaeIII or EcoRI suggesting that a single copy of the L-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish L-FABP cDNA hybridized to a transcript of 700 nucleotides in total zebrafish RNA. In situ hybridization and emulsion autoradiography revealed that the L-FABP was expressed exclusively in the liver of the adult zebrafish. Based on amino acid sequence similarity, the isoelectric point and its tissue-specific pattern of expression, we conclude that this zebrafish FABP belongs to the basic liver-type FABPs only found, thus far, in non-mammalian vertebrates.

Evidence for leptin expression in fishes.

Tissues from bony fish were screened with anti-mouse leptin antibodies to detect the presence of the fat-regulating hormone in fishes. Low molecular-weight (16 kDa) immunoreactive bands were detected in blood, brain, heart and liver of green sunfish (Lepomis cyanellus), bluegill sunfish (Lepomis macrochirus), largemouth bass (Micropterus salmoides), white crappie (Pomonix annularis), channel catfish (Ictalurus punctatus), and rainbow trout (Oncorhynchus mykiss). To further verify that we had identified leptin, the response of fish "leptin" was measured in fed and fasted green sunfish. Fed sunfish had approximately threefold higher concentration of leptin in blood than did fasted sunfish (fed vs. fasted; 0.599 +/- 0.03 microg/microl vs. 0.196 +/- 0.04 microg/microl; P > F = 0.0001), which is consistent with mammalian models of leptin function. Brain leptin concentration is also positively correlated with percent body fat in white crappie and bluegill. Based upon electrophoretic mobility, immunoreactivity, response to fasting, and correlation with adiposity, we believe we have the first evidence for leptin expression in an ectotherm.

Phylogenetic Sequence Analysis, Recombinant Expression, and Tissue Distribution of a Channel Catfish Estrogen Receptor β

An estrogen receptor β (ERβ) cDNA fragment was amplified by RT-PCR of total RNA extracted from liver and ovary of immature channel catfish. This cDNA fragment was used to screen an ovarian cDNA library made from an immature female fish. A clone was obtained that contained an open reading frame encoding a 575-amino-acid protein with a deduced molecular weight of 63.9 kDa. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of channel catfish ERβ on the basis of 25 full-length teleost and tetrapod ER sequences. The consensus tree obtained indicated the existence of two major vertebrate ER subtypes, α and β. Within each subtype, and in accordance with established phylogenetic relationships, teleost and tetrapod ER were monophyletic confirming the results of a previous analysis (Z. Xia et al., 1999, Gen. Comp. Endocrinol. 113, 360–368). Extracts of COS-7 cells transfected with channel catfish ERβ cDNA bound estrogen with high affinity (Kd = 0.21 nM) and specificity. The affinity of channel catfish ERβ for estrogen was higher than previously reported for channel catfish ERα. As determined by qualitative RT-PCR, the tissue distributions of ERα and ERβ were similar but not identical. Both ER subtypes were present in ovary and testis. ERα was found in all other tissues examined from juvenile and mature fish of both sexes. ERβ was also found in most tissues except, in most cases, whole blood and head kidney. Interestingly, the pattern of expression of ER subtypes in head kidney always corresponded to the pattern in whole blood. In conclusion, we isolated a channel catfish ERβ with ligand-binding affinity and tissue expression patterns different from ERα. Also, we confirmed the validity of our previously proposed general classification scheme for vertebrate ER into α and β subtypes and within each subtype, into teleost and tetrapod clades.

Purification and Characterization of Hepatic and Intestinal Phenol Sulfotransferase with High Affinity for Benzo[a]pyrene Phenols from Channel Catfish, Ictalurus punctatus

Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP–agarose affinity columns. SDS–PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with Km values in the 40–100 nM range and Vmax 125–300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 μM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17β-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.