Phenol sulfotransferase in humans: properties, regulation, and function

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitters. All human tissues that have been studied in detail contain at least two forms of PST. One form is thermolabile (TL), catalyzes the sulfate conjugation of micromolar concentrations of dopamine and other phenolic monoamines, and is relatively resistant to inhibition by 2,6-dichloro-4-nitrophenol (DCNP). The other form is thermostable (TS), catalyzes the sulfate conjugation of micromolar concentrations of simple phenols such as p-nitrophenol, and is relatively sensitive to DCNP inhibition. These two forms of PST have been physically separated and partially purified from several human tissues, including an easily accessible tissue, the blood platelet. The biochemical properties of platelet PST are very similar to those of PST in human brain, liver, and small intestine. Individual differences in the basal activity of TS PST in the platelet are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex (r = .94, n = 15, P less than 0.001). In addition, both platelet TS and TL PST activities are correlated significantly with the extent of sulfate conjugation of orally administered drugs such as acetaminophen and methyldopa. These latter observations are compatible with the conclusions that platelet PST activity may reflect the activity of the enzyme at sites of drug metabolism, and that variation in PST activity is one factor responsible for individual differences in the sulfate conjugation of orally administered drugs.     

Human Phenol Sulfotransferase: Correlation of Brain and Platelet Activities

Phenol sulfotransferase (PST; EC 2.8.2.1) catalyzes the sulfate conjugation of phenolic and catechol neurotransmitters and drugs. The human blood platelet has been the most thoroughly studied source of PST because of the possibility that the regulation of the enzyme in this easily accessible tissue might reflect the regulation of PST in the CNS. The human brain and platelet contain at least two forms of PST, forms designated as thermostable (TS) and thermolabile (TL) PST. TS PST catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol and TL PST catalyzes the sulfate conjugation of dopamine and other monoamines. This study was performed to determine whether individual variations in the activities of human platelet TS and TL PST reflect individual variations in cerebral cortical PST activities. PST activities were measured in platelets and in cerebral cortical tissue obtained from 15 patients with epilepsy during clinically indicated neurosurgery. There was a highly significant correlation between the activities of the TS form of PST in cerebral cortex and platelets of these patients (r = 0.940, p less than 0.001), but there was not a significant correlation between activities of the TL form of PST in the two tissues (r = 0.396, p greater than 0.14). In addition to variations in the level of enzyme activity, there are also wide individual variations in the thermal stability of platelet TS PST.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.     

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.     

Genetic Polymorphism for Human Platelet Thermostable Phenol Sulfotransferase (Ts Pst) Activity

Platelet TS PST basal activity and thermal stability were measured in blood samples from 237 individuals in 50 nuclear families. Significant correlations were found among first degree relatives, confirming the previously reported familial aggregation of TS PST basal activity and thermal stability. Commingling analysis of basal TS PST activity provided evidence for multiple component distributions, and after transformation to remove skewness, segregation analysis supported a major gene hypothesis. For TS PST thermal stability, commingling analysis also provided evidence for multiple component distributions. However, segregation analyses were equivocal with regard to the presence of a major gene for thermal stability, since support for a major gene model depended on skewness. Bivariate commingling analysis, which examined thermal stability by simultaneously considering basal activity and activity after heating, suggested that genotypes, as defined by the inferred component distributions for TS PST activity, differ in thermal stability. A three-allele model is proposed as one hypothesis that may account for the combined results of basal activity and thermal stability. The results of this study indicate that a major gene polymorphism in conjunction with polygenic inheritance plays an important role in the regulation of both level of activity and thermal stability of this important drug-metabolizing enzyme in humans.     

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.     

Formation of bile acid glucosides and dolichyl phosphoglucose by microsomal glucosyltransferases in liver, kidney and intestine of man

Formation of glucosides of the bile acids chenodeoxycholic, ursodeoxycholic, deoxycholic and hyodeoxycholic acids has been detected in microsomes from human liver, kidney and intestinal mucosa. Hepatic and extrahepatic bile acid glucosyltransferase activities were characterized with respect to kinetic parameters and other catalytic properties. Whereas no marked organ-specific differences in the affinities of individual bile acids toward hepatic and extrahepatic glucosyltransferases were observed, microsomes from extrahepatic sources showed twice to 5-times the maximal rates of bile acid glucosidation estimated with microsomes from liver. In addition to bile acid glucoside formation, microsomes from human liver, kidney and intestinal mucosa catalyzed the synthesis of dolichyl phosphoglucose acting as natural glucosyl donor in bile acid glucosidation.     

A method to analyze allele-specific methylation

We have developed a method to analyze the methylation patterns of individual alleles of a gene. The target gene must have alleles identifiable by restriction fragment length polymorphism analysis. The method involves separation of the alleles after digestion by restriction enzyme digestion and electrophoresis, followed by recovery from the gel on ion-exchange paper. Methylation analysis can be done on the separate alleles by Southern blot after digestion by methylation-sensitive enzymes. As an example, we studied human c-Ha-ras-1 and showed that the methylation patterns of different alleles are stable and inherited. The method can be applied to the study of inheritance and methylation in genes where alleles can be identified by restriction fragment length polymorphism.     

Characterization of a 28-kD polymorphic polypeptide detected by two-dimensional electrophoresis of human platelets.

A genetic polymorphism of a human platelet polypeptide with a molecular weight of 28 kD detected by two-dimensional electrophoresis was investigated in family and population studies, and cell distribution. The 28-kD polypeptide showed autosomal codominant inheritance of two alleles. The gene frequencies of the two alleles were 0.925 and 0.075, respectively. The 28-kD polypeptide was observed in lymphocytes, neutrophils, eosinophils and monocytes, in addition to platelets. This polypeptide showed good reproducibility in electrophoresis, and appears to be useful as a genetic marker of the human genome in gene mapping and pedigree analysis.     

The effects of flavonoids on human phenolsulphotransferases: potential in drug metabolism and chemoprevention

Flavonoids are frequently found in fruits and vegetables, and are currently under investigation as potential chemopreventive agents. The present study reports the inhibitory effects of six flavonoids, quercetin, genistein, daidzein, equol, (+)-catechin and flavone, on sulphation of p-nitrophenol, a model substrate for the P-form of PST (thermostable, TS) and dopamine, a model substrate for the M-form of PST (thermolabile, TL). Kinetic studies of the PST activity and the inhibitory effects of flavonoids on the P-form of PST activity (using Hanes-Wolf and Dixon plots) show low Km and Ki values. Quercetin was found to be the most potent inhibitor and flavone was the least active inhibitor among the flavonoids. Kinetic analysis showed that the inhibition was non-competitive and Ki values were determined for each flavonoid. These observations suggest the potential for clinically important pharmacological and toxicological interactions by flavonoids.     

Family studies of plasma dopamine-beta-hydroxylase thermal stability.

There are large individual variations in the thermal stability of human plasma dopamine-beta-hydroxylase (DBH). These variations are a characteristic of the DBH molecule itself. Individual subjects may be classified as those with thermolabile and those with thermostable plasma DBH. Of 362 randomly selected unrelated children, 8.01%, and of 238 randomly selected unrelated adult subjects, 5.46% had thermolabile plasma DBH. There was not a significant correlation of DBH thermolability with either sex or age on the basis of data from 230 adults and children in 53 randomly selected families. Subjects with thermolabile DBH had basal enzyme activity only about 55% of that in subjects with stable enzyme. There was not a direct relationship between DBH thermolability and the allele DBHL, the presence of which results in very low basal enzyme activity. There was a significant familial aggregation of the trait of DBH thermolability, but there was not a significant correlation of this trait among spouses. Although preliminary pedigree evaluation raised the possibility of monogenic inheritance of the trait of DBH thermolability by an autosomal recessive mechanism, three separate families in which both parents had thermolabile enzyme included offspring with thermostable DBH. All five of these offspring had very low basal plasma DBH and were presumed to be homozygous for the allele DBHL. These observations raised the possibility that the trait of plasma DBH thermolability may be inherited, and that there may be an interaction between the locus or loci responsible for thermal stability and the locus DBH.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.     

Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells

The phase II metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase (PST, EC 2.4.2.1) was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines (e.g., dopamine) as well as most catecholamine metabolites. The affinity (Km) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucuronyltransferase (EC 2.4.1.17) activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.     

Genetic diversity within and among maize populations: a comparison between isozyme and nuclear RFLP loci

 In order to compare the potential of enzyme and DNA markers to investigate genetic diversity within and among populations, ten maize populations were characterized for (1) 20 isozyme loci and (2) restriction fragment length polymorphism (RFLP) for 35 probe-enzyme combinations. Each population was represented by a sample of at least 30 individuals. The average number of alleles detected per locus was clearly higher for RFLPs (6.3) than for isozymes (2.4). Similarly, total diversity was higher for RFLPs (0.60) than for isozymes (0.23). This difference is consistent with observations on inbred-line collections and can be related to the fact that many variations at the DNA level do not change the amino-acid composition or the global charge of proteins. By contrast, the magnitude of population differentiation, relative to the total diversity, was similar for isozymes (23%) and RFLPs (22%). This suggests that the isozyme and RFLP loci considered in this study are subject to similar evolutionary forces, and that both are mostly neutral. However, RFLPs proved clearly superior to isozymes both to (1) identify the origin of a given individual and (2) reveal a relevant genetic structure among populations. The higher polymorphism observed for RFLP loci and the greater number of these loci contributed to the superior discriminative ability of the RFLP data. Received: 1 June 1997 / Accepted: 3 November 1997     

The Deduced Amino Acid Sequences of Human Platelet and Frontal Cortex Monoamine Oxidase B Are Identical

Abstract: Monoamine oxidases (MAOs) A and B play important roles in the metabolism of neuroactive, vasoactive amines. Human platelets contain only MAO B, often used as an indicator of brain MAO B. The validity of this model remained to be evaluated. This report describes the molecular cloning of human MAO B from frontal cortex and platelets. Two overlapping PCR-amplified clones of human platelet MAO B and four PCR-amplified clones of human frontal cortex MAO B covering the entire coding region were sequenced using five internal oligomers and M13 reverse and forward primers. The nucleotide sequences of human MAO B cDNA from platelet and frontal cortex were identical to that of human liver MAO B except for three nucleotides that differed in frontal cortex: nucleotides 440 A → G, 794 C → T, and 825 C → T. Whether or not these differences are artifactual, all three represent silent mutations, which would not alter the amino acid of the encoded polypeptides. Thus, the deduced amino acid sequences of MAO B from frontal cortex, platelet, and liver are identical. These findings indicate the validity of using platelet MAO B mRNA as a marker for brain MAO B and provide a new approach to study the role of brain MAO B in humans.     

Erythrocyte isozymes of phosphofructokinase in genetically high- and low-2,3-diphosphoglycerate rats

A major locus (Dpg) with two alleles (d and D) controls erythrocyte 2,3-diphosphoglycerate (DPG) levels in Long-Evans rats and is closely linked to a locus (Hbb) determining a hemoglobin electrophoretic polymorphism. Glycolytic intermediate levels and phosphofructokinase (PFK) kinetic studies suggest that in vivo PFK activity differences underlie the differences in DPG levels. We report here chromatographic and immunologic evidence that rat erythrocyte PFK is composed of two isozymes which elute from DEAE-Sephadex at positions identical to those of the isozymes in platelets and liver, respectively. The percentage of platelet-type PFK is significantly (P less than 0.05) smaller in low-DPG (dd) hemolysates than in DD hemolysates regardless of hemoglobin phenotype. When hemolysates were prepared in a stabilizing buffer, PFK specific activity was significantly (P less than 0.005) higher in DD rats. These data suggest that the PFK kinetic differences may result from alterations in the isozyme composition of active PFK.     

Human red cell acid phosphatase: purification and properties of the A, B and C isozymes

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.     

Chinook salmon NADP+-dependent cytosolic isocitrate dehydrogenase: Electrophoretic and genetic dissection of a complex isozyme system and geographic patterns of variation

Species in the genus Oncorhynchus express complicated isocitrate dehydrogenase (IDHP) isozyme patterns in many tissues. Subcellular localization experiments show that the electrophoretically distinct isozymes of low anodal mobility expressed predominantly in skeletal and heart muscle are mitochondrial forms (mIDHP), while the more anodal, complex isolocus isozyme system predominant in liver and eye is cytosolic (sIDHP). The two loci encoding sIDHP isozymes are considered isoloci because the most common allele at one of these loci cannot be separated electrophoretically from the most common allele of the other. Over 12 electrophoretically detectable alleles are segregating at the two sIDHP* loci in chinook salmon. Careful electrophoretic comparisons of the sIDHP isozyme patterns of muscle, eye, and liver extracts of heterozygotes reveal marked differences between the tissues with regard to both relative isozyme staining and the expression of several common alleles. Presumed single-dose heterozygotes at the sIDHP isolocus isozyme system exhibit approximate 9:6:1 ratios of staining intensity in liver and eye, while they exhibit approximate 1:2:1 ratios in skeletal muscle. The former proportions are consistent with the equal expression of two loci (isolocus expression), while the latter are consistent with the expression of a single locus. Screening of over 10,000 fish from spawning populations and mixed-stock fishery samples revealed that certain variant alleles (*127, *50) are detectable only in liver and eye, while other alleles (*129, *94, and *74) are strongly expressed in muscle, eye, and liver. The simplest explanation for these observations is that the "isolocus" sIDHP system of chinook salmon (and that of steelhead and rainbow trout) results from the expression of two distinct loci (sIDHP-1* and sIDHP-2*) that have the same common allele (as defined by electrophoretic mobility). IDHP expression in skeletal muscle is due to the nearly exclusive expression of the sIDHP-1* locus, while IDHP expression in eye and liver tissues is due to high levels of expression of both sIDHP-1* and sIDHP-2*--giving rise to the isolocus situation in these latter tissues. Direct inheritance studies confirm this model of two genetically independent (disomic) loci encoding sIDHP in chinook salmon. Extensive geographic surveys of chinook salmon populations from California to British Columbia reveal marked differences in allele frequencies at both sIDHP-1* and sIDHP-2* and considerably more interpopulation differentiation than was recognized previously when sIDHP was treated as an isolocus system with only five recognized alleles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allele-specific methylation of the human c-Ha-ras-1 gene

The methylation status of individual c-Ha-ras-1 alleles in human cells was measured by Hpall/Mspl analysis of a polymorphic (VTR) region. The gene was extensively methylated in leukocytes and sperm, with the former exhibiting a highly specific methylation pattern. Individual ras alleles were differentially methylated at the VTR region in fetal fibroblasts and immortal cell lines. A new polymorphism in the 5'-flanking region of the gene was also detected. The presence or absence of an Xhol site showed a striking and complete concordance with the length of the VTR region and suggested that the site had been lost by a previous allele-specific mutation.