Diane M. Otterness

Sulfation and sulfotransferases 1: Sulfotransferase molecular biology: cDNAs and genes.

Sulfotransferase (ST) enzymes cata‐lyze the sulfate conjugation of many hormones, neu‐rotransmitters, drugs, and xenobiotic compounds. These reactions result in enhanced renal excretion of the sulfate‐conjugated reaction products, but they can also lead to the formation of “bioactivated” metabolites. ST enzymes are members of an emerging gene superfamily that presently includes phenol ST (PST), hydroxysteroid ST (HSST), and, in plants, flavonol ST (FST) “families,” members of which share at least 45% amino acid sequence iden‐tity. These families can be further subdivided into “subfamilies” that are at least 60% identical in amino acid sequence. For example, the PST family includes both PST and estrogen ST (EST) subfamilies. Amino acid sequence motifs exist within ST enzymes that are conserved throughout phylogeny. These signature sequences may be involved in the binding of 3 ‘‐phosphoadenosine‐5 ‘‐phosphosulfate, the cosubstrate for the sulfonation reaction. There are presently five known human cytosolic ST en‐zymes: an EST, an HSST, and three PSTs. cDNAs and genes for all of these enzymes have been cloned, and chromosomal localizations have been reported for all five genes. Genes for these human enzymes, as well as those of other mammalian cytosolic ST enzymes that have been cloned, show a high degree of structural homology, with conservation of the lo‐cations of most intron/exon splice junctions. Human ST enzyme expression varies among individuals. Functionally significant genetic polymorphisms for ST enzymes in humans have been reported, and other molecular genetic mechanisms that might be involved in the regulation of the expression of these enzymes are being explored. Knowledge of the mo‐lecular biology of cytosolic ST enzymes, when placed within a context provided by decades of biochemical research, promises to significantly enhance our understanding of the regulation of the sulfate conjugation of hormones, neurotransmitters, and drugs.—Weinshilboum, R. M., Otterness, D. M., Aksoy, I. A., Wood, T. C., Her, C., Rafto‐ gianis, R. B. Sulfotransferase molecular biology: cDNAs and genes. FASEB J. 11, 3‐14 (1997)

Human thiopurine methyltransferase pharmacogenetics: Gene sequence polymorphisms

Thiopurine methyltransferase (TPMT) catalyzes the S‐methylation of thiopurine drugs. TPMT activity is regulated by a common genetic polymorphism that is associated with large individual variations in thiopurine toxicity and efficacy. We previously cloned the functional gene for human TPMT and reported a common variant allele for low enzyme activity, TPMT*3A, that contains point mutations at cDNA nucleotides 460 and 719. In the present study, we set out to determine the number, types, and frequencies of TPMT variant alleles associated with low enzyme activity in clinical laboratory samples in the United States and to compare those results with data obtained from two different ethnic groups. We identified a total of six different variant alleles for low TPMT activity in the 283 clinical laboratory samples studied. The most common variant was *3A; the second most frequent variant allele, *3C, contained only the nucleotide 719 polymorphism; and four other variant alleles were detected. TPMT*3A also appeared to be the most common variant allele in a Norwegian white population sample, but it was not found in a population sample of Korean children. However, *3C was present in samples from the Korean children, as was a novel allele, *6. Characterization of variant alleles for low TPMT enzyme activity will help make it possible to assess the potential clinical utility of deoxyribonucleic acid‐based diagnostic tests for determining TPMT genotype.

Olsalazine and 6-mercaptopurine-related bone marrow suppression : A possible drug-drug interaction

A patient with refractory Crohns disease had two separate episodes of bone marrow suppression while receiving 50 to 75 mg 6‐mercaptopurine a day and 1000 to 1750 mg olsalazine a day. This adverse reaction necessitated dose reduction of 6‐mercaptopurine on the first occasion and withdrawal of 6‐mercaptopurine and olsalazine on the second occasion. The patients red blood cell thiopurine methyltransferase (TPMT) activity was 12.2 U per milliliter red blood cells (low normal range) and her TPMT genotype was wild‐type sequence for all known alleles of TPMT that result in low TPMT enzyme activity. In vitro enzyme kinetic studies confirmed the hypothesis that olsalazine and olsalazine‐O‐sulfate are potent noncompetitive inhibitors of recombinant human TPMT. We suggest that the patients relatively low baseline level of TPMT activity was inhibited by olsalazine and olsalazine‐O‐sulfate, leading to decreased clearance of 6‐mercaptopurine and its accumulation. This ultimately increased intracellular 6‐thiopurine nucleotide levels to toxic concentrations, which caused bone marrow suppression.

Purine substrates for human thiopurine methyltransferase

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). A genetic polymorphism regulating TPMT activity in human tissue is an important factor responsible for individual differences in the toxicity and therapeutic efficacy of these drugs. Because of the clinical importance of this polymorphism, we studied 18 purine derivatives, including ribonucleosides and ribonucleotides, as potential substrates for purified human kidney TPMT. Sixteen of the compounds studied were substrates for the enzyme, with Km values that varied from 29.1 to 1270 microM and with Vmax values that varied from 75 to 2340 U/mg protein. The thiopurines tested had Km values that were uniformly lower than were those of the corresponding ribonucleosides or ribonucleotides. 6-Selenopurine derivatives had the lowest Km values of the compounds studied. Finally, oxidized purines with an OH in the 8-position were methylated by the enzyme, but 2-OH compounds were potent inhibitors of TPMT.

Dehydroepiandrosterone sulfotransferase gene (STD): localization to human chromosome band 19q13.3

Dehydroepiandrosterone (DHEA) sulfotransferase (ST) catalyzes the sulfate conjugation of DHEA and other steroid compounds. The human gene for DHEA ST (STD) was mapped by the polymerase chain reaction to chromosome 19 using human x rodent somatic cell hybrid panels. Fluorescence in situ hybridization was then used to localize the STD gene to the region 19q13.3.

Thiopurine methyltransferase regulation in rat kidney: Immunoprecipitation studies

1. Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT activity in the kidneys of male Sprague-Dawley (S-D) rats is approximately twice that present in the kidneys of female S-D rats, and this difference is testosterone-dependent. Renal TPMT activities in these animals also increase dramatically during growth and development. 2. Our studies were conducted to determine whether variations in TMPT activity in the S-D rat kidney were due to differences in the quantity of TPMT protein. Rabbit polyclonal antibodies to partially purified rat kidney TPMT were used to develop an immunoprecipitation assay for immunoreactive TPMT protein. 3. Gender-related differences in renal TPMT activities in S-D rats were due to a lower content of immunoreactive TPMT protein in kidneys of female animals. TPMT enzyme activities and immunoreactive protein levels were also directly correlated in renal preparations from castrated and sham-operated male rats, from testosterone-treated castrated and sham-operated male rats, and from testosterone-treated and control female rats. 4. There was also a significant positive correlation between TPMT enzymic activities and immunoreactive TPMT protein levels in renal tissue from different aged male S-D rats (rs = 0.955, n = 15, P less than 0.001.) 5. These results demonstrate that changes in S-D kidney TPMT activity during growth and development, in the two sexes and in response to testosterone, were due to variations in the quantity of immunoreactive TPMT protein.