Forms Of Phenol Sulfotransferase Research Articles

Mechanism of posttranslational regulation of phenol sulfotransferase: expression of two enzyme forms through redox modification and nucleotide binding.

Sulfotransferase catalyzes sulfuryl group transfer between a nucleotide and a variety of nucleophiles that may be sugar, protein, xenobiotics, and other small molecules. Nucleotides may serve as cosubstrate, cofactor, inhibitor, or regulator in an enzyme catalyzed sulfuryl group transfer reaction. We are trying to understand how nucleotide regulates the activity of phenol sulfotransferase (PST) through the expression of two enzyme forms. The homogeneous rat recombinant PST was obtained from Escherichia coli, and the nucleotide copurified was examined. The nucleotide was completely removed from inactive PST in high salt and oxidative condition. Total enzyme activity was recovered following incubation in reductive environment. Many nucleotides are known to tightly bind to PST but only one nucleotide, 3'-phosphoadenosine 5'-phosphate (PAP), was identified to combine with PST by ion-pair RP-HPLC, UV-visible spectra, (31)P NMR, and ESI-MS and MS-MS spectrometry. In addition to the presence or absence of PAP, oxidation following reduction of PST was required to completely interconvert the two forms of PST. According to the experimental results, a mechanism for the formation of the two enzyme forms was proposed.

Bacterial Expression and Functional Characterization of a Rat Thyroid Hormone Sulfotransferase, ST1B1

At least three forms of phenol sulfotransferase (ST) ST1B1, ST1A1 and ST1C1 are contained in rat livers. To identify the form contributing to the metabolism of 3,3',5-triiodothyronine (T3), functional characterization of these forms was performed by expression in Escherichia coli. ST1B1 and ST1C1 were shown to be active on sulfation towards T3 with high affinity (Km: 44.4 and 25.8 [iM, respectively), whereas ST1A1 had low affinity. In Western blotting using antibodies raised against the individual ST, hepatic contents of each ST were quantitatively determined. ST1B1 showed no clear sex-difference, whereas the level of ST1C1 was higher in adult males than adult females. The content of ST1B1 was 1.4, 6.8 and 10 times higher than that of ST1C1 in adult males, adult females and both sexes of immature rats, respectively. The developmental pattern of ST1B1 was similar to that of ST1A1, but differed from that of ST1C1. These results indicate that ST1B1 and ST1C1 are involved in T3 metabolism in rats and ST1B1 is the constitutive form across sexes and ages.

Effects of 3′-phosphoadenosine 5′-phosphate on the activity and folding of phenol sulfotransferase

Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase. Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction. The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction. Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP. Differences between two forms of phenol sulfotransferase, α and β, indicate that they assemble through different folding process. It is demonstrated that only α enzyme renatures in the presence of PAP and β enzyme renatures only in the absence of PAP in vitro. In the over-expressed system, formation of α and β phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli. It is concluded that folding of phenol sulfotransferase is assisted by PAP to form α enzyme. In the absence of PAP, β form of phenol sulfotransferase is produced.

Human Phenol SulfotransferaseSTP2Gene: Molecular Cloning, Structural Characterization, and Chromosomal Localization

Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of “simple” planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs,STP2,as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans.STP2spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of mostSTP2exon–intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans,STM;a rat PST gene; a human estrogen ST (EST) gene,STE;and a guinea pig EST gene. The two initialSTP2exons, IA and IB, were identified by performing 5′-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5′-untranslated region sequences. The two apparent 5′-flanking regions of theSTP2gene, regions flanking exons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements.STP2was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization ofSTP2will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissue.

Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone.

During the secretory phase of the human menstrual cycle, the endometrium is minimally responsive to the estrogens secreted from the ovaries. Conjugation of beta-estradiol (E2) with sulfate is thought to be an important mechanism in the regulation of the levels of active E2 in endometrial tissue. Estrogen sulfation is reportedly increased during the secretory phase in response to the high levels of progesterone secreted by the ovaries. Estrogen sulfotransferase (hEST), a distinct form of human cytosolic sulfotransferase (ST) with an affinity for E2 and estrone at low nanomolar concentrations, has recently been cloned and expressed in mammalian cells and in bacteria (J Steroid Biochem Mol Biol 52:529, 1995). At least two other forms of human cytosolic ST, dehydroepiandrosterone ST (hDHEA-ST) and the phenol-sulfating form of phenol-ST (hP-PST), also conjugate estrogens but at micromolar concentrations. This report describes the specific induction of hEST in human Ishikawa endometrial adenocarcinoma cells by progesterone as a model for the increases in estrogen sulfation observed in women during the secretory phase of the menstrual cycle. Treatment of Ishikawa cells with 10 microns progesterone for 48 h resulted in a 7-fold increase in the sulfation of 20 nM E2. The sulfation of selective substrates for human dehydroepiandrosterone sulfotransferase (hDHEA-ST) and the two forms of phenol sulfotransferase (hP-PST, hM-PST) were not affected by treatment with progesterone. The levels of immunoreactive hEST and hEST mRNA in the Ishikawa cells were both increased by progesterone, whereas the levels of immunoreactive hDHEA-ST, hP-PST, and hM-PST were not altered. hEST activity was not induced by treatment of Ishikawa cells with varying concentrations of E2, testosterone, or cortisol. The induction of hEST by progesterone was inhibited by RU-486, indicating that progesterone is acting via the progesterone receptor. These results indicate that progesterone is capable of specifically inducing hEST and estrogen sulfation in human Ishikawa adenocarcinoma cells and suggest a mechanism for increasing estrogen sulfation in the endometrium during the secretory phase of the menstrual cycle.

Molecular Cloning of a Novel Form of Phenol Sulfotransferase from Human Brain Hippocampus

Sulfate conjugation by sulfotransferases is important for the metabolism of drugs, xenobiotics, monoamines, and neuropeptides (1-3). In this study, molecular cloning resulted in the isolation of a 1.2-kb human brain hippocampal cDNA encoding a member of the phenol sulfotransferase (PST) family of enzymes. The hippocampal phenol sulfotransferase (H-PST) cDNA possesses differences in its deduced primary sequence compared to human liver monoamine (M form) (4,5) and phenol (P form) (6,7) isoforms of PST. The hippocampal PST contrasts with previous studies that identified two human brain PST cDNAs whose coding sequences are identical to the human liver M and P forms of PST. Importantly, this study provides evidence for a human hippocampal form Of PST.

Changes in substrate specificity of the recombinant form of phenol sulfotransferase IV (tyrosine-ester sulfotransferase)

The over-expression of mammalian enzymes in bacterial systems by means of recombinant DNA technology has provided the enzymologist with a supply of catalyst sufficiently abundant to identify suboptimal substrates. Such large quantities are particularly useful when working with the enzymes of detoxication, a family of proteins that are distinguished by their broad substrate specificity for generally lipophilic compounds, i.e., by their very low specificity for features other than the functional group [1]. We have achieved bountiful expression of a sulfotransferase active with phenols [2], an enzyme originally purified and characterized from rat liver [3], and classified as tyrosine-ester sulfotransferase, EC 2.8.2.9 [4,5], but usually referred to as rat liver phenol or aryl sulfotransferase IV. Having improved the sensitivity and versatility of some of the assays for sulfotransferases, we examined the substrate spectrum of this enzyme. As presented here, the results of this examination point to the limitations of enzyme nomenclature and to the danger of equating enzymes isolated from their normal habitat with those formed by recombinant technology in a foreign cell. Our experiments also establish a greater catalytic scope for the natural rat liver enzyme than that previously described.

Human Liver Estrogen Sulfotransferase: Identification by cDNA Cloning and Expression

Sulfation is an important pathway in the biotransformation of steroid hormones such as estrogens. Human liver contains three well-characterized sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Although two of these enzymes, DHEA ST and one form of PST, can catalyze the sulfate conjugation of estrogens, our goal was to test the hypothesis that human liver might also contain a distinct estrogen ST (EST). We used the PCR to clone a human liver EST cDNA by utilizing degenerate oligonucleotide primers designed on the basis of highly conserved EST sequences in non-human species to amplify a 512 nucleotide portion of the open reading frame (ORF) with single-stranded human liver cDNA as a template. Rapid amplification of cDNA ends (RACE) was then used to obtain the 5′- and 3′-ends of the EST cDNA. The ORF of this cDNA consisted of 882 nucleotides and encoded 294 amino acids. The protein encoded by the human liver EST cDNA was 81%, 73%, and 72% identical to the amino acid sequences of guinea pig adrenocortical, bovine placental and rat liver ESTs, respectively. Human liver EST transiently expressed in COS-1 cells was capable of catalyzing the sulfation of estrone, DHEA and 4-nitrophenol, but not that of dopamine. The expressed human liver EST was also characterized with regard to thermal stability, inhibition by 2,6-dichloro-4-nitrophenol (DCNP) and response to NaCl. Identification of human liver EST by cloning and expression of its cDNA should enhance understanding of the enzymology and regulation of the biotransformation of estrogens and other steroids in humans.

Human Liver Thermolabile Phenol Sulfotransferase: cDNA Cloning, Expression and Characterization

The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PSI). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids -- including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, alpha-methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (alpha-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated alpha-methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic, intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP35S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (Km 5 to 50 microM) and a low affinity reaction (Km 360 to 2,900 microM). The efficiency of sulfation (Vmax/Km) for both reactions was 5-fold higher for (+)- than for (-)-ISO. When the hepatic PSTs were resolved by ion-exchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a Vmax/Km value 6.1-fold higher for (+)- than for (-)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a Vmax/Km value 5.7-fold higher for (+)- than for (-)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (-)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation.

An HPLC-ECD procedure for measuring total phenolsulfotransferase (PST) activity in human liver, platelets and blood

The phenolsulfotransferase (PST) activity in human liver, platelets and blood was measured under saturating concentrations of the conjugating agent, 3'-phospho-adenosine-5'-phosphosulfate (PAPS). Conventional PST assays employ PAP 35S at suboptimal concentrations. In addition, the sulfate conjugate formed, namely N-acetyldopamine-sulfate (NADA-sulfate) was quantified directly by high-pressure liquid chromatography cum electrochemical detection (HPLC-ECD). NADA, the biogenic amine acceptor used in this study appeared from kinetic data to be a substrate of both the P and M forms of PST when used in micromolar concentration. Two apparent K m values of 4.2 μmol/l and 22.6 μmol/l were observed. In contrast, only one apparent K m value was evident when the assay was carried out in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a selective inhibitor of the P form of PST or after heat treatment under specified conditions which inactivates the M form of PST. Thus measurement of PST activity with NADA as the acceptor substrate permits the determination of total PST activity and a parallel assay with the inclusion of DCNP would distinguish the two variants of PST, both of which appear to be present in all human tissues.

Sulfation pharmacogenetics: correlation of human platelet and small intestinal phenol sulfotransferase.

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs. All human tissues studied contain a thermostable (TS) form of PST, which catalyzes the sulfate conjugation of "simple" phenols such as p-nitrophenol, and a thermolabile (TL) form, which catalyzes the sulfation of dopamine and other monoamines. In the present study we tested the hypothesis that genetically controlled levels of TS and TL PST activity in the platelet, as well as inherited variations in the thermal stability of platelet TS PST, might reflect those same characteristics of the enzyme in the less accessible tissue, human small intestinal mucosa. Platelet TS and TL PST activities and TS PST thermal stability were measured in blood samples from 45 randomly selected healthy subjects, and 14 of those subjects were selected to have intestinal biopsies performed. There was a significant correlation between levels of platelet and jejunal mucosal TS PST activity (rs = 0.574, p less than 0.030), but there was not a significant correlation between levels of TL PST activity in the two tissues (rs = 0.265, p = 0.368). There was also a significant correlation between the trait of TS PST thermal stability in the two tissues (rs = 0.828, p less than 0.0001). These observations suggest that inherited variations in TS PST activity and thermal stability in an easily obtained tissue, the platelet, might be used to predict individual differences of those properties of the enzyme in the human small intestine, an organ that plays an important role in drug metabolism.

Triiodothyronine: A Substrate for the Thermostable and Thermolabile Forms of Human Phenol Sulfotransferase*

The enzyme(s) responsible for the sulfate conjugation of L-T3 in man has not been characterized. T3 sulfotransferase (T3-ST) activity was characterized in normal human liver tissue obtained during clinically indicated surgical resection. Subcellular distribution studies showed that the T3-ST activity was localized to the cytoplasmic fraction. This finding raised the possibility that T3-ST activity might be similar to the 2 previously identified forms of cytoplasmic phenol sulfotransferase (PST) found in human tissue. A thermostable (TS) form of PST catalyzes the sulfate conjugation of micromolar concentrations of p-nitrophenol, and a thermolabile (TL) form catalyzes the sulfate conjugation of dopamine and other monoamines. Thermal stability and enzyme inhibitor experiments showed that T3-ST activity in pooled liver homogenates was very similar to the TS form of PST. The apparent similarity of T3-ST to TS PST was studied further by measuring T3-ST, TS PST, and TL PST activities in 20 individual liver samples. T3-ST activities correlated significantly with TS PST activities (r = 0.939; P less than 0.001) measured with p-nitrophenol, but not with TL PST activities (r = -0.118; P greater than 0.6) measured with dopamine. However, sulfation of T3 by the TL form of the enzyme might have been masked by the 18-fold higher specific activity of TS than TL PST in human liver homogenates. When the two forms of PST were separated by ion exchange chromatography, T3 was found to be a substrate for both the TS and TL forms of PST. "True" Km values for T3 were similar for TS and TL PST (81 and 127 microM, respectively).

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside G M2

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of G M2 or G M2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped α-[ 14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by G M2 Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of G M2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[ 3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.

Phenol sulfotransferase inheritance.

1. Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic and catechol neurotransmitters. Human tissues contain both thermostable (TS) and thermolabile (TL) forms of PST that differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. 2. Individual variations in the levels of activity of both TS and TL PST in the human platelet are strongly influenced by inheritance. 3. Individual differences in the level of platelet TS PST activity are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex, liver, and intestinal mucosa. 4. There are also individual familial differences in the thermal stability of TS PST in the platelet. These differences are correlated with individual variations in the thermal stability of TS PST in cerebral cortex, liver, and intestinal mucosa. 5. Individual variations in the thermal stability of TS PST in hepatic tissue are associated with the presence of one or both of a pair of TS PST isozymes that can be separated by ion-exchange chromatography and that differ in their thermal stabilities. 6. This series of observations suggests that a structural gene polymorphism may be one mechanism by which inheritance controls TS PST in humans. The isozymes of TS PST in liver may represent the products of alternative alleles for this polymorphism, alleles that might control the structure of TS PST in many human tissues.

Human liver phenol sulfotransferase: Assay conditions, biochemical properties and partial purification of isozymes of the thermostable form

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitiers. Human platelets and brain contain at least two forms of PST. One form is relatively thermolabile (TL) and catalyzes the sulfate conjugation of monoamines such as dopamine. The other is thermostable (TS) and catalyzes the sulfation of “simple” phenols such as phenol and p-nitrophenol. We found that homogenates of human liver also contain two forms of PST that are similar to brain and platelet TL and TS PST with regard to substrate specificities, thermal stabilities and sensitivities to inhibitors. Optimal conditions were determined for the assay of these two activities in human liver homogenates. The apparent K m of liver homogenate TL PST for dopamine was 27 μM. The apparent K m of the TS form of the enzyme for p-nitrophenol was 0.94 μM. Human liver TS PST also catalyzed the sulfate conjugation of dopamine, but with an apparent K m of 5 mM, over two orders of magnitude higher than that of TL PST. Two different peaks of TS PST activity were separated from the TL activity by ion exchange chromatography of human liver preparations. Both peaks of TS PST activity were partially purified and characterized. Both had similar substrate specificities and inhibitor sensitivities. K m values of TS PST peak I for p-nitrophenol and for 3'-phosphoadenosine-5'-phosphosulfate were 0.91 and 0.86 μM, respectively, while the K m values of TS PST peak II for these two cosubstrates for the reaction were 0.43 and 0.64 μM, respectively. However, the TS PST activity in peak II was significantly more thermolabile than was the activity in peak I. These results are compatible with the conclusion that human liver homogenates contain at least two forms of PST, forms with properties similar to those of TS and TL PST in homogenates of human cerebral cortex and platelets. In addition, human liver contains two isozymes of TS PST.

Inhibition of M and P phenol sulfotransferase by analogues of 3'-phosphoadenosine-5'-phosphosulfate.

Structural analogues of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (3',5'-PAPS) were examined for their ability to inhibit dopamine and phenol sulfation by the M and P forms of phenol sulfotransferase (PST), respectively. The Ki values for each of the adenosine derivatives were calculated from the rate equation for PST. For both M and P PST, the naturally occurring product 3'-phosphoadenosine-5'-phosphate, (3',5'-PAP), was shown to be the most effective inhibitor. The weakest inhibitors of the two sulfotransferases were 5'-adenosine phosphosulfate and the three AMP derivatives, which were less than 1,000 times as effective as 3',5'-PAP. 5'-ATP, 2',5'-PAPS, 2',5'-PAP, and 5'-ADP were similar in their inhibition of M and P PST and were all approximately 100 times less effective than the natural end product. These data reveal that there is a rigid structural requirement for binding of the ribose portion of adenosine to both M and P PST that involves the groups on both the 3' and 5' positions. The effectiveness of binding to the two enzymes may depend on both steric factors as well as the distribution of negative charges on the ribose ring.

Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P I-PST) was sub-sequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P I-PST proceeds via a sequential ordered, bisubstrate reaction mechanism, where 3′-phosphoadenosine-5′-phosphosulfate (PAPS) is the leading substrate. The true K m and K i a values for PAPS were both 0.35 μ m, while the true K m value for phenol was 2.8 μ m.

Human platelet phenol sulfotransferase: familial variation in thermal stability of the TS form.

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catechol and phenolic drugs and xenobiotic compounds. Platelets and other tissues contain at least two forms of PST, forms that have been designated the "TL" and the "TS" forms. We measured the thermal stability of platelet TS PST in blood samples from 218 randomly selected unrelated subjects by heating platelet homogenates at 44 degrees C for 15 min. Thermal stability was expressed as the ratio of the enzyme activity remaining after preincubation to that in an unheated sample, a heated/control (H/C) ratio. The frequency distribution of H/C ratios for this population sample was bimodal, with a nadir at an H/C ratio of 0.33. Of the 218 subjects studied, 29 (13.3%) had thermolabile TS PST (H/C less than 0.33). Platelet samples were then obtained from subjects with thermolabile and thermostable TS platelet PST. PST activity in these platelet samples had similar apparent Km constants for substrates. IC50 values for inhibition of TS PST by 2,6-dichloro-4-nitrophenol in these samples were also nearly identical. The results of experiments in which platelet homogenates from subjects with thermolabile and thermostable TS PST were mixed and the results of experiments in which platelet homogenates were subjected to gel filtration chromatography were compatible with the conclusion that individual differences in TS PST thermal stability were properties of PST itself. Finally, there was a significant familial aggregation of the trait of thermolabile TS PST when H/C ratios were measured in platelet homogenates from 231 members of 49 randomly selected families.