New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines

Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.     

A simplified radioenzymatic assay for dihydrofolate reductase using [3H]dihydrofolate

This report describes a simple method to measure the activity of dihydrofolate reductase using the substrate [³H]dihydrofolate, which is generated by preincubation of [³H]folic acid for 10 min with dithionite before the enzymatic reaction. The procedure then measures the direct reduction of [³H]dihydrofolate to [³H]tetrahydrofolate by coprecipitating the unreduced substrate with excess unlabeled folic acid and acidified zinc sulfate. The advantage of this method is that [³H]dihydrofolate, which is not commercially available, can be generated from high specific activity [³H]folic acid, which is commercially available, immediately before initiating the enzymatic reaction. By this modification, the two important advantages of radioenzymatic assays for dihydrofolate reductase can be more easily exploited; namely, increased sensitivity because much less substrate need be used, and the ability to measure enzyme activity in crude tissue preparations without interference by precipitating proteins or nucleotide oxidases.     

Formation of [Leu5]Enkephalin from Dynorphin A(1–8) by Rat Central Nervous Tissue In Vitro

[3H]Dynorphin A(1-8) is readily metabolised by rat lumbosacral spinal cord tissue in vitro, affording a variety of products including a significant amount (20% recovered activity) of [3H][Leu5]enkephalin. In the presence of the peptidase inhibitors bestatin, captopril, thiorphan, and leucyl-leucine, [3H][Leu5]enkephalin was the major metabolic product, accounting for 60% of recovered activity. Production of [3H][Leu5]enkephalin was seen across all gross brain regions. The enzyme responsible for the cleavage has an optimal substrate length of 8-13 amino acids and is inhibited by N-[1-(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a site-directed inhibitor of the metalloendopeptidase EC 3.4.24.15. However the enzymic breakdown also has properties in common with involvement of endo-oligopeptidase A. Possible consequences of the formation of [Leu5]-enkephalin from the smaller dynorphins are discussed.     

Heterogeneity of high affinity uptake of L-glutamate and L-aspartate in the mammalian central nervous system.

Characteristics of high affinity uptake of L-glutamate are examined in order to evaluate the possible use of the uptake of [3H]L-glutamate, [3H]L-aspartate or any other suitable [3H]-labelled substrate as a marker for glutamatergic and aspartergic synapses in autoradiographic studies in the mammalian brain. Review of data on substrate specificity indicates the presence of at least two high affinity uptake systems specific for acidic amino acids in the central nervous tissue; one which takes up L-glutamate and L-aspartate and the other which is selective for L-glutamate only. Studies on ionic requirements, too, point to the existence of at least two distinct uptake systems with high affinity for L-glutamate. The Na(+)-dependent uptake system(s) handle(s) both L-glutamate and L-aspartate whereas the Na(+)-independent uptake system(s) show(s) selectivity for L-glutamate only. Available data do not favour the Na(+)-dependent binding of [3H]D-aspartate to thaw-mounted sections of frozen brain tissue as a suitable marker for glutamatergic/aspartergic synaptic nerve endings. However, there are reasons--such as the results of lesion studies and the existence of uptake sites which have a higher affinity for L-aspartate than for D-aspartate--to suggest that Na(+)-dependent binding of [3H]L-aspartate, rather than that of [3H]D-aspartate, should be further investigated as a possible marker for the glutamatergic/aspartergic synapses in the autoradiographic studies using sections of frozen brain.     

3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.     

Phosphatidic Acid and Phosphoinositide Turnover in Myelin and Its Stimulation by Acetylcholine

Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phosphatidate (PA). Distribution of the 32P-labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5-fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol-containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20-60% stimulation of [32P]phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C-dependent cycle relative to de novo biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid radioassay for sphingosine kinase

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A–II. MEASUREMENT OF THE TURNOVER RATES OF MAO A DURING ONTOGENESIS IN THE RAT BRAIN

The combined measurement of MAO A activity (using [³H]5-HT as a specific substrate) and [³H]harmaline binding capacity indicated that the concentration of MAO A in brain was higher in 14-28 day old rats than in adult animals. The turnover rates of this enzyme in the forebrain and the brain stem of young (14-28 day old) and adult rats were calculated by following the recovery of MAO A activity and of [³H]harmaline binding capacity after an acute treatment with pargyline (75mg/kg i.p.). Both the fractional rate constant for MAO A degradation and its synthesis rate per g of fresh tissue were significantly higher in young animals. However, the calculation of the absolute synthesis rates of MAO A per brain area gave very similar values in young and adult animals: 1.3-1.5 × 10¹³ molecules of MAO A synthesized per day in the forebrain and 2.3-2.9 × 10¹² molecules per day in the brain stern. The results illustrate the validity of using [³H]harmaline binding to evaluate possible changes in the turnover rate of MAO A in tissues.     

L-[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.     

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5-4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala-kindled and "yoked" control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of gamma-aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.     

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.     

Na+ + K+] ATP-ase in liver and brain of obese mice

The activity of hepatic [Na+ + K+]ATP-ase showed a gene-dosage relationship in 6 week old mice. Before weaning hepatic [Na+ + K+]ATP-ase activity was normal in preobese mice but fell within 7 days of weaning to the low levels observed in older ob/ob mice. Brain [Na+ + K+]ATP-ase activity was unchanged in ob/ob mice although [3H]-ouabain binding was reduced. Arrhenius plots of [Na+ + K+]ATP-ase activity in liver and brain and of [3H]-ouabain binding to brain preparations showed breakpoints at lower temperatures in ob/ob than lean mice. These breakpoints were altered by pretreatment of tissue with deoxycholate. It is suggested that changes in membrane lipid composition might be an important factor regulating [Na+ + K+]ATP-ase in ob/ob mice.     

A New Synaptosomal Biosynthetic Pathway of Glutamate and GABA from Ornithine and Its Negative Feedback Inhibition by GABA

In sonicates of mouse brain synaptosomes, we demonstrated that gamma-aminobutyric acid (GABA) can be formed when L-ornithine (Orn) through L-glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]L-proline (Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1-5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine delta-aminotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30-fold-purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, L-glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or beta-alanine. The inhibition of GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Orn is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of L-glutamic-gamma-semialdehyde in equilibrium with L-delta 1-pyrroline-5-carboxylic acid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It is confirmed that L-canaline (delta-aminooxy-L-alpha-aminobutyric acid) is a potent and specific inhibitor of brain OAT whereas much weaker inhibition was observed with two other carbonyl-trapping agents, aminooxyacetic acid and hydrazine.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Affinity States of Rat Brain Opioid Receptors in Different Tissue Preparations

The binding of [3H]Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET), selective agonists for mu- and delta-opioid binding sites, respectively, has been investigated using different rat brain tissue preparations and buffer systems. The results were compared with the binding of the ligands to crude membrane fractions in Tris-HCl, the most commonly used preparation for binding studies. In both rat brain membranes and intact cells, Krebs-HEPES induced a decrease in the affinities of [3H]DAGO and [3H]DTLET, but little modification was observed when 20-microns tissue slices were used, whatever the brain area studied. The dissociation rate of [3H]DTLET was clearly dependent on the tissue preparation used, because the koff value of this ligand in Krebs-HEPES was 2.5-fold higher in membrane fractions than that measured in intact cells. The kinetic dissociation constant of [3H]DTLET in membrane fractions in Krebs-HEPES was 6.5-fold greater than that measured in Tris-HCl. In intact cells, the koff value for [3H]DTLET was lower than that found in membrane fractions in Krebs-HEPES and similar to that observed in membrane preparations in Tris-HCl supplemented with 30 mM NaCl. These data suggest (a) that the koff constant of [3H]DTLET was regulated by the ionic environment of the delta-opioid receptor, which is clearly dependent on the preservation of cellular structure, and (b) that opioid receptors could exist under different states that are regulated, in part, by the intracellular Na+ concentration.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]gamma-vinyl-GABA to GABA-transaminase in brain homogenates

[3H]Gamma-vinyl-GABA, an irreversible inhibitor of GABA-transaminase, was used to label the enzyme in homogenates of rat brain. The binding procedure utilized was found to be specific for GABA-transaminase and linear with tissue obtained from several regions of rat brain up to concentrations of 8 micrograms protein/microliter. The specific binding was directly proportional to the activity of the enzyme measured in vitro and was completely inhibited by the GABA-transaminase inhibitors aminooxyacetic acid (100 microM) and 3-mercaptopropionic acid (1.0mM). The binding procedure was used to estimate the amount of active enzyme present in a homogenate of striatal tissue.     

Characterization of [3H]Ouabain Binding Sites in Human Brain, Platelet, and Erythrocyte

[3H]Ouabain binding was investigated in membranes prepared from human brain, erythrocyte, and platelet. Scatchard analysis of [3H]ouabain binding to human hypothalamic membranes revealed a single class of noninteracting binding sites with an apparent affinity constant (KD) of 21 nM. Though the number of [3H]ouabain binding sites was lower in human platelets than in erythrocytes, both tissues exhibited a single class of high-affinity binding sites with an apparent KD similar to that found in human brain. Specific [3H]ouabain binding in basal ganglia tissue from patients with Huntington's disease was more than 50% lower than in tissue from age- and sex-matched controls. These results, along with previous findings in rat brain, suggest that high-affinity [3H]ouabain binding labels the neuronal form of Na, K-ATPase in human brain, and may prove useful in quantitating this enzyme in postmortem brain samples.     

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).