Showing papers by "Richard M. Weinshilboum published in 1993"

Human thiopurine methyltransferase : molecular cloning and expression of T84 colon carcinoma cell cDNA

Abstract: Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Levels of TPMT activity in human tissue are controlled by a common genetic polymorphism that is an important factor responsible for individual variation in thiopurine drug toxicity and therapeutic efficacy. Our goal was to purify, to obtain a partial amino acid sequence for, and to clone and express cDNA for human TPMT as a first step in determining the molecular basis for this genetic polymorphism. Human kidney TPMT was purified, the protein was subjected to limited proteolysis, and amino acid sequence information was obtained from the resultant peptide fragments. Primers based on the amino acid sequence information were used to amplify a unique sequence from human liver cDNA by use of the polymerase chain reaction. Because TPMT has been reported to be present in the colon, T84 human colon carcinoma cells were studied and were found to express TPMT activity with biochemical properties similar to those of the human kidney and liver enzymes. Oligonucleotide probes based on the human kidney TPMT amino acid sequence were then used to screen a T84 human colon carcinoma cell cDNA library. A 2.7-kilobase cDNA clone was isolated that contained an open reading frame of 735 nucleotides, which encoded a protein of 245 amino acids. The deduced amino acid sequence of the encoded protein included one 24- and two separate 12-amino acid sequences identical to those obtained by sequencing proteolytic fragments of purified human kidney TPMT. Transcripts were made in vitro from the open reading frame of the cDNA clone. These transcripts were translated in a rabbit reticulocyte lysate system, and the resulting translation product comigrated with human kidney TPMT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T84 cell cDNA clone, truncated within the 3' untranslated region at an Sstl restriction site, was then used to create an expression construct with the eukaryotic expression vector P91023(B), and this construct was used to transfect COS-1 cells. The transfected cells expressed a high level of TPMT enzymatic activity, and this activity displayed a pattern of inhibition by TPMT inhibitors identical to that of human kidney and T84 human colon carcinoma cell TPMT. Cloning of cDNA for this important drug-metabolizing enzyme may make it possible to define the molecular basis of the TPMT genetic polymorphism in humans.

Human liver dehydroepiandrosterone sulfotransferase: Nature and extent of individual variation

Abstract: Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of steroid hormones such as DHEA, estrone, and estradiol. As a first step in pharmacogenetic studies of DHEA ST in humans, we measured individual variation in DHEA ST enzymatic activity and thermal stability in 94 samples of human hepatic tissue, 39 of which were from patients with normal liver function studies. Neither level of enzyme activity nor thermal stability were significantly correlated with either time of tissue storage at -80 degrees C or patient age. In addition, there were no gender-dependent differences in DHEA ST activity in these samples. DHEA ST enzymatic activity varied 4.6-fold, with a mean value of 317 +/- 100 units/gm tissue (mean +/- SD) in all samples and 318 +/- 104 units/gm in the subset of 39 samples from patients with normal hepatic function studies. Frequency distributions of DHEA ST activity for both the entire group of 94 samples and the subset of 39 were bimodal, with 25% and 21% included in a high activity subgroup, respectively. The presence of this high activity subgroup was confirmed when data for samples from male and female patients were evaluated separately and when only data for white patients were examined. The existence of a subgroup of subjects with a high level of DHEA ST enzymatic activity in liver and a 4.6-fold range in this activity have implications for individual differences in the sulfate conjugation of endogenous and exogenously administered steroid hormones and raise the possibility of pharmacogenetic regulation of this important enzyme in humans.

Catechol O-methyltransferase pharmacogenetics: photoaffinity labelling and Western blot analysis of human liver samples

Abstract: The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (M(r)) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]-S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had M(r) values of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either M(r) or pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.

Cholesterol sulfation in human liver. Catalysis by dehydroepiandrosterone sulfotransferase.

Abstract: Cholesterol can undergo sulfate conjugation to form cholesterol 3-sulfate. Our experiments were performed to determine whether human liver cytosol could catalyze the sulfation of cholesterol, and, if so, whether any of the three well-characterized human hepatic cytosolic sulfotransferases, dehydroepiandrosterone sulfotransferase (DHEA ST), thermostable (TS) phenol sulfotransferase (PST), or thermolabile (TL) PST might participate in the reaction. On the basis of substrate kinetics, two "forms" of cholesterol sulfotransferase (CST) activity were present in human liver cytosol, one with high and one with low affinity for cholesterol. Apparent KM values of the high- and low-affinity activities were 0.14 and 15 microM for cholesterol and 0.30 and 0.19 microM for 3'-phosphoadenosine-5'-phosphosulfate, respectively. Both kinetic forms of CST activity had thermal inactivation profiles similar to those of DHEA ST and TS PST, but both were more thermostable than was TL PST. Enzyme inhibition studies performed with 2,6-dichloro-4-nitrophenol (DCNP) showed that inhibition profiles for both high- and low-affinity CST activities were similar to those of DHEA ST and TL PST, but both were more resistant to DCNP inhibition than was TS PST. Experiments performed with 20 individual human liver samples confirmed these observations and demonstrated highly significant correlations between both high- and low-affinity CST activities and DHEA ST activity (rs = 0.740, p = 0.0001 and rs = 0.767, p < 0.0001, respectively). However, the level of activity of neither kinetic form of CST activity was significantly correlated with either TS or TL PST activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Diethyldithiocarbamate S-methylation: evidence for catalysis by human liver thiol methyltransferase and thiopurine methyltransferase.

Abstract: Disulfiram is used in the treatment of alcoholism to inhibit the enzyme aldehyde dehydrogenase. Disulfiram is rapidly reduced in vivo to form diethyldithiocarbamate (DDC), and DDC can undergo methyl conjugation to form S-methyl-DDC. Human tissues contain two separate genetically regulated enzymes that can catalyze thiol S-methylation. Thiol methyltransferase (TMT) is a microsomal enzyme that preferentially catalyzes, the S-methylation of alipathic sulfhydryl compounds, whereas thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds. Our experiments were performed to determine whether human liver microsomal and/or cytosolic preparations could catalyze the S-methylation of DDC, and, if so, to determine whether TMT or TPMT might be the enzymes involved. We found that both human liver microsomes and cytosol could catalyze DDC S-methylation. The microsomal activity displayed biphasic substrate kinetics, with apparent Km values for DDC of 7.9 and 1500 microM for the high- and low-affinity activities, respectively. The high-affinity activity had an apparent Km value for S-adenosyl-L-methionine, the methyl donor for the reaction, of 5.8 microM. The thermal inactivation profile and response to methyltransferase inhibitors of the high-affinity microsomal DDC S-methyltransferase activity were similar to those of human liver microsomal TMT. In addition, TMT activity and the activity catalyzing the S-methylation of DDC were highly correlated in 19 individual liver samples (rs = 0.956; P < .0001). Hepatic cytosolic DDC S-methyltransferase activity had an apparent Km value for DDC of 95 microM. The cytosolic enzyme which catalyzed DDC S-methylation and TPMT activity had similar thermal inactivation profiles, similar patterns of response to methyltransferase inhibitors and the two activities coeluted during ion exchange chromatography. Furthermore, the activities of TPMT and cytosolic DDC S-methyltransferase were highly correlated in 20 individual liver samples (rs = 0.963; P < .0001). These results were compatible with the conclusion that both TMT and TPMT could catalyze the S-methylation of DDC in the human liver. Because the activities of both TMT and TPMT are controlled by inheritance, our observations raise the possibility of pharmacogenetic variation in the biotransformation and therapeutic effect of DDC in humans.

Genetic segregation analysis of red blood cell (RBC) histamine N‐methyltransferase (HNMT) activity

Abstract: Methylation is an important pathway in the biotransformation of many drugs, neurotransmitters, and xenobiotic compounds. Histamine N-methyltransferase (HNMT) catalyzes the Nτ-methylation of histamine and structurally related compounds. Measurement of HNMT activity in the RBC makes it possible to access variation in the enzyme activity that may reflect differences in less accessible tissues such as brain. Previously reported high family correlations for RBC HNMT activity suggested that genetic inheritance plays a major role in the regulation of variation in this enzyme. In the present study we completed complex segregation analyses of RBC HNMT activity of 241 individuals in 51 nuclear families that were randomly ascertained through children in the Rochester, Minnesota public school system in order to characterize the mode of inheritance of this important enzyme. We found evidence for major gene influence on the regulation of RBC HNMT activity. Both transformed and untransformed data support the presence of Mendelian major gene segregation, but the gene frequency differences do not indicate a direct correspondence between genotypes inferred from the two sets of analyses. Analyses of the skewed untransformed data indicated the presence of a relatively rare (Q = 0.121) additive major gene for high activity, with the three overlapping genotype distributions representing 77, 21, and 2 % of individuals. Analyses of the normalized transformed data indicated the presence of a common (Q = 0.71) additive major gene for high activity, with the three overlapping genotype distributions accounting for 9, 41, and 50 % of individuals. The analyses of transformed data give the best fit as well as the most parsimonious Mendelian major gene model. However, we cannot rule out the possibility of multiple alleles, and analyses of untransformed data provide some support for a third allele. Molecular studies will be needed to validate and characterize the alleles that regulate RBC HNMT activity levels in humans. © 1993 Wiley-Liss. Inc.

Histamine N-methyltransferase: inhibition by monoamine oxidase inhibitors.

Abstract: HistamineN-methyltransferase (HNMT) catalyzes theNτ-methylation of histamine.Nτ-Methylhistamine can then undergo oxidation catalyzed by the mitochondrial enzyme monoamine oxidase (MAO). Addition of an MAO inhibitor such as pargyline to tissue preparations can increase the HNMT activity assayed-presumably as a result of inhibition ofNτ-methylhistamine metabolism by MAO. However, pargyline-dependent “activation” of HNMT may also occur in tissue preparations that lack mitochondria. Our experiments were performed to determine whether MAO inhibitors, like many other amine compounds, could directly increase the activity of partially purified HNMT, and, if so, to study the mechanism of activation. Human kidney HNMT was partially purified by sequential ion exchange and gel filtration chromatography. The activity of the purified HNMT was increased approximately 50% in the presence of pargyline. However, enzyme kinetic experiments showed that pargyline, like many other amines, was a competitive inhibitor of HNMT. Apparent activation of the enzyme resulted from sequential shifts of histamine substrate curves to higherV max values in the presence of increasing concentrations of pargyline. Other acetylenic MAO inhibitors, clorgyline and the two stereoisomers of deprenyl, were also competitive inhibitors of purified human kidney HNMT. Inhibition kinetic experiments performed in the presence of varying concentrations of histamine demonstrated thatK is values for pargyline, clorgyline, (R)-deprenyl and (S)-deprenyl were 0.126, 0.144, 0.217, and 0.627 mM, respectively. When the concentration of the cosubstrate for the reaction,S-adenosyl-l-methionine, was varied in the presence of variable concentrations of pargyline, inhibition of HNMT by pargyline was noncompetitive with regard to the methyl donor, withK ii andK is values of 1.23 and 0.95 mM, respectively. Finally, several amine compounds related structurally to pargyline were also found to be inhibitors of HNMT.

Human liver microsomal thiol methyltransferase : inhibition by arylalkylamines

Abstract: 1. Thiol methyltransferase (TMT) is a microsomal enzyme catalyzing the S-methylation of aliphatic sulphydryl drugs and xenobiotics. Studies of the functional significance of S-methylation catalysed by TMT have been hampered by lack of a potent, relatively specific, non-toxic inhibitor of the enzyme.2. Human hepatic microsomal TMT was inhibited by the arylalkylamine 2,3-dichloro-α;-methylbenzylamine (DCMB), and by a series of arylalkylamines, as well as the arylamme, aniline.3. Inhibition kinetic studies with DCMB, benzylamine, aniline, phenylethylamine and phenylethanolamine, five compounds with a wide range of IC50 values, showed ‘mixed’ inhibition of TMT with respect to the methyl acceptor substrate, 2-mercaptoethanol. Kis and Kii values were, respectively, 1.1 and 0.29 μM for DCMB, 160 μM each for benzylamine, 680 and 370 μM for aniline, 1640 and 1380 μM for phenylethylamine, and 2300 and 1400 μM for phenylethanolamine. Inhibition was at least partially reversible.4. H.p.l.c. analyses were carried out ...