Acetylation of p-aminobenzoic acid by human blood
Abstract
Acetylation of p-aminobenzoic acid was studied in human blood cell lysates. The rate of acetylation with acetyl-coenzyme A was 3.4 nmoles per min per 0.5 ml lysate (corresponding to 0.5 ml blood with a 50% hematocrit). In the presence of a coenzyme A generating system, the rate was only 0.1 nmole per min per 0.5 ml lysate. N-Acetyltransferase (acetyl-CoA : arylamine N-acetyltransferase, EC 2.3.1.5) activity exhibited a temperature optimum within the range of 34–37° with a Q10of 2 between 24–34°. Heating for 3 min at 50° caused 50 per cent inactivation of enzymatic activity. The pH activity profile showed an optimum at pH 6.0–6.5. p-Chloromercuribenzoate was a potent inhibitor causing 50 per cent inhibition at 9 μM. The apparent Km value for p-aminobenzoic acid was 0.4 mM and for acetylcoenzyme A, 0.3 mM. The enzyme activity with p-aminosalicyclic acid was about 50 per cent that obtained with p-aminobenzoic acid. Acetylation of sulfamethazine was either very low or in several individuals undetectable. Isonicotinic acid hydrazide at a concentration 10 times that of p-aminobenzoic acid did not interfere with acetylation of the latter, nor did trimethoprim, another compound with an amine moiety. Folic acid and amethopterin competitively inhibited the acetylation of p-aminobenzoic acid, with respective Ki values of 0.01 mM and 0.06 mM. It is concluded that the activity of N-acetyltransferase in human blood may have important clinical implications.
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Acetyl CoA: Arylamine N-Acetyltransferase activity in rat hepatocytes cultured on different extracellular matrices
1997, Toxicology in VitroN-Acetyltransferase (NAT) activity towards p-aminobenzoic acid and sulfamethazine was examined in primary cultures of rat hepatocytes cultured on three extracellular matrices (ECM)—type I collagen, thermally denatured type I collagen, and Matrigel®. Whereas protein and DNA content declined markedly during the first 24 hr of culture, p-acetylamidobenzoate (AcPABA) and N-acetylsulfamethazine (AcSMZ) formation were readily detectable on all three ECM for the 6-day culture period. Protein and DNA content, as well as NAT activities, were higher on Matrigel than on either of the other two ECM. Additional studies were conducted to confirm the expression of both enzymes during the culture period. The ratio of AcPABA to AcSMZ formation remained relatively stable throughout the 6-day culture period, suggesting that both enzymes continued to be expressed throughout the study period. Further studies in cells cultured on Matrigel revealed that AcPABA formation exhibited a time-dependent decline when cytosol from cultured cells was incubated at 50 °C, whereas AcSMZ formation proved to be thermostable. Moreover, methotrexate substantially inhibited AcPABA formation, but had only modest effects on AcSMZ. These studies support the conclusion that AcPABA and AcSMZ are predominantly formed by way of different enzymes throughout the culture period. These findings are supported by the observation that NAT1 and NAT2 mRNA were detectable on all days examined. These data indicate that primary cultures of rat hepatocytes should prove useful in probing the regulation of NAT and its role in toxicity.
Purification of recombinant human N-Acetyltransferase type 1 (NAT1) expressed in E. Coli and characterization of its potential role in folate metabolism
1995, Biochemical PharmacologyHuman arylamine N-acetyltransferase type 1 (NAT1) has been cloned from human genomic DNA, into the vector pET(5a) and expressed in Escherichia coli. The recombinant protein has been purified to apparent homogeneity using anion exchange chromatography. The arylamine acceptor specificity, and the effect of potential NAT1 inhibitors has been investigated using purified recombinant protein. The Km of the recombinant NAT1 protein for the substrates para-aminobenzoate (p-aba) and 4-aminosalicylate are 14.3 and 11.8 μM, respectively. Folate and amethopterin were found to be potent competitive inhibitors of p-aba acetylation, with Ki values of 13.3 and 9.5 μM, respectively. The pteroate moiety of folate, in contrast is a poor inhibitor, with 100 μM pteroate inhibiting only 40% of NAT1 activity. A catabolite of folate para-aminobenzoly-l-glutamate has also been shown to be a NAT1 substrate with a Km value of 263 μM.
Identification of a substance, previously shown to enhance mitogenesis of human lymphocytes, as the acetamide of P-aminobenzoic acid
1994, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid MetabolismWe characterize here an arachidonic acid (AA)-derived metabolite previously found to have an adjuvant effect in phytohemagglutinin-induced mitogenesis of lymphocytes from mothers of newborn babies and from immunodeficient infants. We named the metabolite ‘compound 4’ due to its position in a thin-layer chromatography system developed for isolation of eicosanoids. The compound was originally found to be produced by peripheral blood mononuclear leukocytes and the T cell leukemia line Jurcat after long-term (18–24 h) incubation with [1-14C]AA. Compound 4 is also produced by lymphocytes, monocytes, platelets, trombocytes, cultured fibroblasts and various types of malignant cell lines. We purified this metabolite by means of high pressure liquid chromatography with synchronous detection of radioactivity and measurement of ultraviolet-light absorption at 278 nm. Proton nuclear magnetic resonance spectroscopy and mass spectrometry with electron impact techniques demonstrated that compound 4 is not an eicosanoid, but is identical to p-acetamidobenzoic acid (PACBA). The cells synthezise PACBA from p-aminobenzoic acid and a two-carbon residue from AA.
Arylamine N-acetyltransferase in human red blood cells
1992, Biochemical PharmacologyN-Acetyltransferase activities associated with erythrocytes from 20 individuals have been determined with p-aminobenzoic acid as substrate. A three-fold variation in Vmax is found. The N-acetyltransferase genotype of the individuals has been determined and there is no correlation between the extent of acetylation measured in the individuals' erythrocytes and the inheritance of alleles at the polymorphic NAT locus. Folate is confirmed to be an inhibitor of arylamine N-acetyltransferase activity measured in erythrocytes. The content of folate in erythrocytes of individuals also varies. The individual with the maximum folate content has the minimum N-acetyltransferase activity. The monomorphic N-acetyltransferase gene from individuals spanning the range of N-acetyltransferase activity have been amplified, using the polymerase chain reaction. The pattern of restriction enzyme digestion of the monomorphic N-acetyltransferase gene with a series of eight restriction enzymes is the same for individuals spanning the activity range of arylamine N-acetyltransferase in their erythrocytes.
In vitro studies on the deacetylation-reacetylation of arylamides and the transacetylation of arylamines by human and rat whole blood
1991, Biochemical PharmacologyHuman and rat whole blood were shown to metabolize the aromatic amides acetanilide and phenacetin by deacetylation followed by reacetylation in vitro. Derivatives of the parent compounds labelled with deuterium in the N-acetyl group produced non-labelled material after incubation. The reaction was monitored by capillary gas chromatographic-mass spectrometric (GC-MS) analysis. There was no significant difference in the acetyl group exchange of these substrates using blood samples donated by non-diabetic volunteers or Type 2 diabetic patients (respective mean ± SEM values = 4.0 ± 0.2% and 4.2 ± 0.3% for trideuteroacetanilide, 6.2 ± 0.6% and 6.1 ±0.3% for trideuterophenacetin). Increasing the glucose concentration in the incubation medium by 50 mmol/L significantly (P < 0.01) increased deacetylation-reacetylation of trideuteroacetanilide in each group (4.6 ± 0.2% and 4.7 ± 0.2% for non-diabetic and diabetic subjects, respectively). In rat blood the amount of deacetylation-reacetylation was much higher: 7.2 ± 0.6% and 8.3 ± 0.7% for trideuteroacetanilide and trideuterophenacetin, respectively. Induction of experimental diabetes using streptozotocin did not significantly change the extent of deacetylation-reacetylation of either deuterated substrate (10.1 ± 2.1% and 9.5 ± 1.1%). Elevation of the incubation glucose concentration by 50 mmol/L produced an increase in acetyl group exchange (for trideuteroacetanilide) in diabetic (14.3 ± 2.2%) and non-diabetic (10.6 ± 1.0%) rats. The donation of acetyl groups (transacetylation) was observed after incubation of blood samples from both diabetic and non-diabetic human subjects and rats with trideuterophenacetin and a molar excess of aniline. This reaction significantly (P < 0.001) decreased the acetyl group exchange of trideuterophenacetin (these values were 4.5 ± 0.4% and 3.4 ± 0.6% using samples from non-diabetic human subjects and rats, respectively) and demonstrated the ability of whole blood to catalyse transacetylation (acetyl-CoA-independent acetylation). There was correlation between the amount of (unlabelled) acetanilide produced by acetylation with acetyl-CoA and the percentage present as trideuteroacetanilide. The proportion of trideuteroacetanilide was higher using rat blood (e.g. the values for non-diabetic subjects were 25.5 ± 1.7% vs 8.5 ± 0.3%; P < 0.001) although the total amount of acetanilide produced was lower (0.54 ± 0.14 nmol vs 1.82 ± 0- 0.23 nmol; P < 0.05) than that observed using human blood.
Inter-individual variation of human blood N-acetyltransferase activity in vitro
1988, Biochemical PharmacologyInter-individual variation in the in vitro acetylation of the antibacterial drug sulphamethazine by human whole blood was studied using reverse phase HPLC. The mean (range) values of blood N-acetyltransferase activity in vitro were 0.50 (0.29–0.83) nmol per 109 red blood cells (rbc) (N = 23), 3.33 (2.22–5.27) nmol per 109 rbc (N = 27) and 9.36 (6.72–15.76) nmol per 109 rbc (N = 23) at initial sulphamethazine concentrations of 0.018 mM, O.18 mM and 1.44 mM respectively. The mean (range) values of the initial rate of sulphamethazine acetylation at these substrate concentrations were 28.1 (20.9–35.0) per 109 rbc (N = 11), 0.26 (0.18–0.42) per 109 rbc (N = 19) and 0.91 (0.61–1.50) per 109 rbc (N = 14) respectively. The mean (range) half life of thermal inactivation of blood acetylation capacity at 50° was 0.91 (0.59–1.27) min (N = 12) at an initial substrate concentration of 0.18 mM. In each of these cases, there was no significant differences between the values obtained using blood samples from rapid and slow acetylators.
Intra-individual variation of blood N-acetyltransferase activity was studied in a single subject on 24 separate occasions during a two year period and was less than 10% at each of the three sulphamethazine concentrations studied. The correlation between the in vitro blood N-acetyltransferase activity of eight volunteers measured on two separate occasions at least 6 weeks apart was 0.84, 0.98 and 0.98 at initial sulphamethazine concentrations of 0.018 mM, 0.18 mM and 1.44 mM respectively.
Increasing the acetyl-CoA concentration of blood samples from 4 subjects by 0.34, 0.85 and 1.67 mM significantly increased both the initial acetylation rate of sulphamethazine and the amount of acetylsulphamethazine produced after an incubation time of 24 hr (initial sulphamethazine concentration = 0.18 mM).