A novel surgical strategy for the resection of duodenal gastrointestinal stromal tumours located close to the duodenal ampulla: a case report.

Although the optimal surgical procedure for the resection of duodenal gastrointestinal stromal tumours has not yet been characterised due to the low prevalence of these tumours and the anatomical complexity of the duodenopancreatic region, difficult surgical procedures such as pancreaticoduodenectomy are often proposed for stromal tumours located in the second portion of the duodenum. Our case report highlights a novel surgical strategy that can be implemented as an alternative to pancreaticoduodenectomy for such tumours close to the duodenal ampulla. A 70-year-old man incidentally diagnosed with a stromal tumour close to the duodenal ampulla in the second portion of the duodenum underwent local resection guided by an endoscopic nasobiliary drainage tube with primary closure. This tube was converted to a percutaneous trans-small intestinal biliary drainage tube during the procedure to prevent biliary leakage biliary stasis due to swelling of the duodenal ampulla. He also underwent a simple distal gastrectomy with Roux-en-Y reconstruction. This resulted in successful R0 resection. There were no procedure-related complications or post-surgery weight changes. Our simple novel surgical strategy may therefore be useful for avoiding pancreaticoduodenectomy and maintaining quality of life in patients with stromal tumours close to the duodenal ampulla.

[Concomitant lobectomy for lung cancer under intraaortic balloon pumping and coronary bypass grafting].

A 71-year-old man was admitted because of an abnormal shadow on the chest X-ray film. Chest computed tomography (CT) revealed a tumor in the right upper lobe. The diagnosis of lung cancer was made by transbronchial lung biopsy. He had suffered an infarction of the inferior myocardial wall at the age of 55 years. Preoperative coronary angiography revealed total occlusion of segment 1, 75% stenosis of segments 5 and 6, and 90% stenosis of segment 13. Since these coronary lesions could cause perioperative and postoperative myocardial infarction, the patient was scheduled to undergo surgery of both the heart and lung in a one-stage operation. Under intraaortic balloon pumping (IABP), we performed a right upper lobectomy of the lung, and coronary artery bypass grafting with cardiopulmonary bypass through median sternotomy. During the lobectomy and first postoperative day, a stable circulation was achieved with IABP. The postoperative course was uneventful. At present, that is 33 months after the operation, the patient presents no sighs of recurrence of lung cancer and has not suffered any anginal attack during follow-up. Lung cancer and coronary artery disease can be treated simultaneously by this procedure.

A 12(S)-hydroxyeicosatetraenoic acid receptor interacts with steroid receptor coactivator-1.

Lewis lung carcinoma cells contain specific high-affinity binding sites for the eicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE]. These binding sites have a cytosolic/nuclear localization and contain the heat shock proteins hsp70 and hsp90 as components of a high molecular weight cytosolic binding complex. The ligand binding subunit of this complex is a protein with an apparent molecular mass of approximately 50 kDa as judged by gel permeation chromatography. In this report, we present data showing that the 50-kDa 12(S)-HETE binding protein interacts as a homodimer with steroid receptor coactivator-1 (SRC-1) in the presence of 12(S)-HETE. Two putative interaction domains were mapped. One of these (amino acids 701-781) was within the nuclear receptor interaction domain in SRC-1 required for binding of various steroid and thyroid hormone receptors. It contains the most C-terminal of the three copies of LXXLL motif present in the nuclear receptor interaction domain. The second interaction domain was present in the N-terminal part of SRC-1 (amino acids 1-221). This region has two LXXLL motifs, one does not bind and the other binds only weakly to steroid and thyroid hormone receptors. Glutathione S-transferase (GST) pulldown experiments and far Western analyses demonstrated that the N-terminal region of SRC-1 (amino acids 1-212) alone does not bind the 50-kDa 12(S)-HETE binding protein, whereas GST/DeltaSRC-1(1-1138) ligand-dependently pulled down a protein of approximately 50 kDa in size. Our results suggest that the 50-kDa 12(S)-HETE binding protein is a receptor that may signal through interaction with a nuclear receptor coactivator protein.

Prostate selectivity of JTH-601-G1, an active metabolite of JTH-601, in dogs.

OBJECTIVE: To evaluate the effect of JTH-601-G1, an active metabolite and glucuronide conjugate of JTH-601 (an alpha1-adrenoceptor antagonist), on smooth muscle contraction in canine prostate and artery, and to examine the effect of JTH-601-G1 on prostatic urethral pressure and blood pressure in anaesthetized dogs. Materials and methods Male beagle dogs were used in both an in vitro and an in vivo study. In the former, the prostate and right common carotid artery were isolated, and smooth muscle strips from the prostate and open-ring strips from the carotid artery prepared. The effects of JTH-601-G1 on phenylephrine- and noradrenaline-induced contraction were assessed in these tissues. In the in vivo study, four dogs were anaesthetized and the change in urethral pressure, blood pressure and heart rate measured continuously. Vehicle (saline) and JTH-601-G1 were then infused intravenously in increasing doses (0.33-3.3 microg/kg/min for 30 min). In three other dogs, the effect of JTH-601-G1 infusion at a higher rate (25 microg/kg/min for 3 h) on blood pressure was evaluated, and the plasma concentration of JTH-601-G1 measured using high-performance liquid chromatography-mass spectrometry. RESULTS: Of the distinct metabolites of JTH-601, JTH-601-G1 had the most potent alpha1-adrenoceptor antagonistic effect in isolated canine prostate. JTH-601-G1 also antagonized alpha1-adrenoceptor agonist-induced contraction in common carotid artery, but the pA2 value in the artery was approximately 25 times higher than that in the prostate. In anaesthetized dogs, JTH-601-G1 decreased urethral pressure in a dose-dependent manner; at the highest dose, urethral pressure decreased by 24.5% and blood pressure by 7.0%. However, there was no significant change in heart rate at any dose. The plasma concentration of JTH-601-G1 increased with the dose of JTH-601-G1, but the concentration of both JTH-601 and other metabolites was below the detection limit. The higher JTH-601-G1 infusion rate caused blood pressure to decrease by only 6-10% even at JTH-601-G1 plasma concentrations of approximately 1500 ng/mL during the infusion. Although there was a negative correlation between mean blood pressure and plasma JTH-601-G1 concentration, the decrease in blood pressure was small compared with the reduction in urethral pressure. CONCLUSION: JTH-601-G1 appears to be a major active metabolite of JTH-601 but with a higher selectivity for canine prostate than artery. The results also indicate that in addition to the alpha1A-adrenoceptor, the alpha1L-adrenoceptor plays an important prostatic selective role in smooth muscle contraction via the alpha1-adrenoceptor.

Inhibition of neuronal nitric oxide synthase by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.

Anandamide amidohydrolase of porcine brain: cDNA cloning, functional expression and site-directed mutagenesis(1).

Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabinoid receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine brain, and the cDNA encoded a protein of 579 amino acids with a molecular mass of 62.9 kDa. The amino acid sequence was 81, 80 and 85% identical with the enzymes previously cloned from the liver of rat, mouse, and human, respectively. When the enzyme protein was overexpressed in COS-7 cells, the particulate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidonic acid and ethanolamine both with a specific activity of 0. 2-0.3 micromol/min/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate specificity hydrolyzing oleamide, 2-arachidonoylglycerol, and methyl arachidonate. The point mutation of Ser-217, Asp-237, Ser-241, or Cys-249 completely abolished the hydrolyses of all the above-mentioned substrates as well as the synthesis of anandamide in the reverse reaction.

Enzymological and molecular biological studies on anandamide amidohydrolase.

Previously we suggested that anandamide amidohydrolase partially purified from porcine brain catalyzed the anandamide synthesis. The reversibility of the anandamide hydrolytic reaction was confirmed with a recombinant enzyme of rat liver. We also showed that the recombinant enzyme had a wide substrate specificity hydrolyzing primary amides and esters of fatty acids in addition to anandamide. When the organ distribution of anandamide amidohydrolase was examined with rats, a large amount of the enzyme was contained in small intestine as well as liver and brain. The intestinal hydrolase was masked by endogenous lipid inhibitors. The enzyme was also found in various eye tissues.

A hydrolase enzyme inactivating endogenous ligands for cannabinoid receptors.

Cannabinoids are psychoactive components of marijuana, and bind to specific G protein-coupled receptors in the brain and other mammalian tissues. Anandamide (arachidonoylethanolamide) was discovered as an endogenous agonist for the cannabinoid receptors. Hydrolysis of anandamide to arachidonic acid and ethanolamine results in the loss of its biological activities. The enzyme responsible for this hydrolysis was solubilized, partially purified from the microsomes of porcine brain, and referred to as anandamide amidohydrolase. In addition to the anandamide hydrolysis, the enzyme preparation catalyzed anandamide synthesis by the condensation of arachidonic acid with ethanolamine. Several lines of enzymological evidence suggested that a single enzyme catalyzes both the hydrolysis and synthesis of anandamide. This reversibility was confirmed by the use of a recombinant enzyme of rat liver overexpressed in COS-7 cells. However, in consideration of the high Km value for ethanolamine as a substrate for the anandamide synthesis, the enzyme was presumed to act as a hydrolase rather than a synthase under physiological conditions. The recombinant enzyme acted not only as an amidase hydrolyzing anandamide and other fatty acid amides but also as an esterase hydrolyzing methyl ester of arachidonic acid. 2-Arachidonoylglycerol, which was found recently to be another endogenous ligand, was also efficiently hydrolyzed by the esterase activity of the same enzyme. The anandamide hydrolase and synthase activities were detected in a variety of rat organs, and liver showed by far the highest activities. A high anandamide hydrolase activity was also detected in small intestine but only after the homogenate was precipitated with acetone to remove endogenous lipids inhibiting the enzyme activity. The distribution of mRNA of the enzyme was in agreement with that of the enzyme activity.

Cryogenic X-ray crystal structure analysis for the complex of scytalone dehydratase of a rice blast fungus and its tight-binding inhibitor, carpropamid: the structural basis of tight-binding inhibition.

Scytalone dehydratase is a member of the group of enzymes involved in fungal melanin biosynthesis in a phytopathogenic fungus, Pyricularia oryzae, which causes rice blast disease. Carpropamid [(1RS,3SR)-2, 2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropa necarboxamide] is a tight-binding inhibitor of the enzyme. To clarify the structural basis for tight-binding inhibition, the crystal structure of the enzyme complexed with carpropamid was analyzed using diffraction data collected at 100 K. The structural model was refined to a crystallographic R-factor of 0.180 against reflections up to a resolution of 2.1 A. Carpropamid was bound in a hydrophobic cavity of the enzyme. Three types of interactions appeared to contribute to the binding. (i) A hydrogen bond was formed between a chloride atom in the dichloromethylethylcyclopropane ring of carpropamid and Asn-131 of the enzyme. (ii) The (chlorophenyl)ethyl group of carpropamid built strong contacts with Val-75, and this group further formed a cluster of aromatic rings together with four aromatic residues in the enzyme (Tyr-50, Phe-53, Phe-158, and Phe-162). (iii) Two hydration water molecules bound to the carboxamide group of carpropamid, and they were further hydrogen-bonded to Tyr-30, Tyr-50, His-85, and His-110. As a result of interactions between carpropamid and the phenylalanine residues (Phe-158 and Phe-162) in the C-terminal region of the enzyme, the C-terminal region completely covered the inhibitor, ensuring its localization in the cavity.

Decreased activity of arachidonate 12-lipoxygenase in platelets of Japanese patients with non-insulin-dependent diabetes mellitus.

To study the metabolism of the platelet 12-lipoxygenase pathway in diabetes, we evaluated the correlation between the activity and amount of arachidonate 12-lipoxygenase in the platelets of patients with non-insulin-dependent-diabetes mellitus (NIDDM). There were four parts in this investigation: (1) examination of abnormalities in platelet 12-lipoxygenase in patients with NIDDM recruited from the Hospital of Juntendo University School of Medicine; (2) comparison of 12-lipoxygenase in the platelets of non-obese NIDDM patients without angiopathy versus normal subjects matched for age, sex, and body mass index (BMI); (3) evaluation of gender differences; and (4) assessment of the potential influence of glycemic control. The activity of 12-lipoxygenase was assayed by incubation of [1-14C]arachidonic acid with the platelet cytosol. The reaction mixture was extracted and separated by thin-layer chromatography, and the radioactive end products were detected. The activity of 12-lipoxygenase in the platelets of patients with NIDDM was significantly less than in normal subjects (P < .003), whereas the amount of 12-lipoxygenase protein did not differ between the two groups. Thus, the specific activity of 12-lipoxygenase in diabetic patients was significantly less than that of normal subjects (P < .001). The enzyme activity and the specific enzyme activity of 12-lipoxygenase in non-obese NIDDM patients without angiopathy were significantly lower than the values in normal subjects matched for gender, age, and BMI (P < .006 and P < .0007, respectively). No significant difference in the activity or amount of platelet 12-lipoxygenase was observed between males and females matched for age, BMI, and disease. In addition, no relationship was observed between 12-lipoxygenase activity and blood glucose levels measured at the time of specimen collection. However, slight negative correlations were noted between 12-lipoxygenase activity and 1,5-anhydroglucitol, hemoglobin A1c (HbA1c), and fructosamine (r = .369, -.354, and -.279, respectively). When recombinant 12-lipoxygenase was incubated with varying concentrations of glucose or fructose, enzyme inactivation was related to the length of incubation, and was unaffected by glucose or fructose. These observations suggest that the activity of 12-lipoxygenase in the platelets of patients with NIDDM is decreased by prolonged hyperglycemia. The mechanism involved requires further investigation.

Distribution of anandamide amidohydrolase in rat tissues with special reference to small intestine.

Anandamide (arachidonylethanolamide), an endogenous ligand for cannabinoid receptors, is hydrolyzed by an amidohydrolase and its biological activity is lost. Previously, we partially purified the enzyme from porcine brain and anandamide synthesis by its reverse reaction was proposed (Ueda et al., (1995) J. Biol. Chem. 270, 23823-23827). The anandamide hydrolase and synthase activities were examined with various rat tissues. Rat liver showed the highest specific activities (4.4 +/- 0.3 and 4.5 +/- 0.5 nmol/min/mg protein) for the hydrolase and synthase, respectively. In most other tissues such as brain, testis and parotid gland, the ratio of synthase/hydrolase activity was 0.7-1.6. However, small intestine showed a relatively high synthase/hydrolase ratio of about 5.0 (1.0 +/- 0.1 and 0.2 +/- 0.1 nmol/min/mg protein). When a homogenate of small intestine was subjected to acetone extraction to remove lipids, a higher hydrolase activity was found (2.0 +/- 0.2 nmol/min/mg protein). Furthermore, Northern blotting detected an intense mRNA band of anandamide hydrolase in small intestine as well as liver and brain. These results demonstrated for the first time a high content of anandamide hydrolase in small intestine.

Reversible hydrolysis and synthesis of anandamide demonstrated by recombinant rat fatty-acid amide hydrolase.

Previously we suggested that one porcine brain enzyme (anandamide amidohydrolase) catalyzed both the hydrolysis of anandamide and its synthesis from arachidonic acid and ethanolamine (Ueda et al., J. Biol. Chem. 270, 23823-23827, 1995). In the present study we investigated the reversibility of the enzyme reactions by the use of recombinant fatty-acid amide hydrolase of rat liver, which appears to be catalytically identical to porcine anandamide amidohydrolase. The particulate fraction of the COS-7 cells, in which the rat enzyme was overexpressed, hydrolyzed anandamide with a specific activity of 132 nmol/min/mg protein at 37 degrees C, and the Km value for anandamide was 18 microM. The enzyme also synthesized anandamide at a rate of 177 nmol/min/mg protein, and the Km values for arachidonic acid and ethanolamine as substrates were as high as 190 microM and 36 mM, respectively. The control cells transfected with the insert-free vector showed neither the hydrolase activity nor the synthase activity. Thus, the hydrolase and synthase are attributed to the same enzyme protein coded by one gene. However, the enzyme may act as a hydrolase rather than a synthase under physiological conditions judging from its high Km values for substrates in the synthase reactions. In addition, primary amides of fatty acids such as arachidonamide and oleamide and fatty acid ester like methyl arachidonate were hydrolyzed at considerable rates, and their reverse reactions occurred even if at lower rates.

Calcium-dependent inactivation of neuronal nitric oxide synthase: evidence for the existence of stabilization / activation factor.

Neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) activity in supernatant of rat cerebellum homogenate was unstable and chelating reagent protected the activity from the rapid decrease. The main target ion of the chelating reagent was found to be Ca2+. Although the enzyme was very unstable after purification by the procedures including DEAE-cellulose chromatography and ammonium sulfate precipitation, the inactivation was neither accelerated by addition of Ca2+ nor protected by EGTA. Upon addition of boiled supernatant of rat cerebellum homogenate, this purified enzyme became more active and stable, but rapid inactivation occurred again by addition of Ca2+, suggesting the existence of previously unreported Ca2(+)-dependent stabilizer / activator in the boiled supernatant. This factor was concentrated by organic solvent and the effects on the enzyme were completely canceled by addition of Ca2+ or phospholipase C treatment.

Metabolism of anandamide, an endogenous cannabinoid receptor ligand, in porcine ocular tissues.

Anandamide (arachidonylethanolamide) is an endogenous ligand for cannabinoid receptors, and exerts various cannabimimetic activities. Since cannabinoids and anandamide were pharmacologically active with the eye, we examined metabolism of anandamide in a variety of porcine ocular tissues. In the presence of ethanolamine, [14C]arachidonic acid was converted to [14C]anandamide by a homogenate of retina, choroid, iris, optic nerve and lacrimal gland with a specific enzyme activity of 1.9-4.2 nmol min-1 mg-1 protein at 37 degrees C. On the other hand, [14C]anandamide was hydrolysed to [14C]arachidonic acid by a homogenate of each tissue with a specific enzyme activity of 1.2-3.5 nmol min-1 mg-1 protein. Thus, both activities of anandamide synthase and hydrolase were found in these ocular tissues. As for the subcellular distribution, the two enzyme activities were mostly recovered in particulate fractions rather than the cytosol. With the retina microsome palmitic acid was converted to its ethanolamide at a lower rate than arachidonic acid, and palmitoylethanolamide was less active than anandamide as a substrate for the hydrolase.

Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase.

Mammalian brain as well as mouse neuroblastoma (N18TG2) and rat basophilic leukaemia (RBL) cells were previously shown to contain "anandamide amidohydrolase', a membrane-bound enzyme sensitive to serine and cysteine protease inhibitors and catalyzing the hydrolysis of the endogenous cannabimimetic metabolite, anandamide (arachidonoyl-ethanolamide). With the aim of developing novel inhibitors of this enzyme, we synthesized three arachidonic acid (AA) analogues, i.e. arachidonoyl-diazo-methyl-ketone (ADMK), ara-chidonoyl-chloro-methyl-ketone (ACMK) and O-acetyl-arachidonoyl-hydroxamate (AcAHA), by adding to the fatty acid moiety three functional groups previously used to synthesize irreversible inhibitors of serine and cysteine proteases. The three compounds were purified and characterized by proton nuclear magnetic resonance and electron impact mass spectrometry. Their effect was tested on anandamide amidohydrolase partially purified from N18TG2 and RBL-1 cells and porcine brain. Pre-treatment of the enzyme with each compound produced a significant inhibition, with ADMK being the most potent (IC50 = 3, 2 and 6 microM) and AcAHA the weakest (IC50 = 34, 15 and 25 microM) inhibitors. The inactivated enzyme regained its full activity when chromatographed by anion-exchange chromatography, suggesting that none of the compounds inhibited the amidohydrolase in a covalent manner. Accordingly, Lineweaver-Burk profiles showed competitive inhibition by each compound. Conversely, the irreversible inhibitor of cytosolic phospholipase As, methyl-arachidonoyl-fluoro-phosphonate (MAFP), covalently inhibited the amidohydrolase. MAFP was active at concentrations 10(3) times lower than those reported for phospholipase A2 inhibition, and is the most potent anandamide amidohydrolase inhibitor so far described (IC50 = 1-3 nM). MAFP, ADMK and ACMK, probably by inhibiting anandamide degradation, produced an apparent increase of the in vitro formation of anandamide from its biosynthetic precursor N-arachidonoyl-phosphatidyl-ethanolamine.

Enzymes for anandamide biosynthesis and metabolism.

Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify "anandamide amidohydrolase' which hydrolyzes anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of porcine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 mumol/min per mg protein (37 degrees C). Apparent K(m) for anandamide was about 60 microM. The enzyme reacted also with ethanolamides of linoleic, oleic, and palmitic acids at lower rates. This enzyme preparation also converted arachidonic acid to anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the anandamide synthase and amidohydrolase activities were derived from a single enzyme protein.

Dietary control of lactase expression in the weaning rat.

The decline in lactase activity during weaning has been well established. However, its molecular mechanism remains to be explored. We studied changes in the expression of lactase in terms of the transcription and translation processes in rat microvillus membrane by Northern blot and Western blot analysis, respectively. To examine the effect of dietary change from a milk to a non-milk diet on the developmental pattern of lactase expression, weaning was prevented by keeping the rats under suckling conditions for 27 days after birth. This treatment only suppressed the extent of decline: while the weanlings showed 17 percent activity compared to that of 4-day-old rats, the prolonged suckling rats showed only 42 percent. The changes in the expression of lactase mRNA and protein were parallel with the change of lactase activity. In other words, the fundamental pattern of significant depression of lactase expression occurred relatively independent of dietary modification. This observation indicates that the regulation of lactase expression is firmly determined at the transcriptional level, and that dietary factor such as the termination of lactose ingestion has only a relatively minor effect.

Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide.

Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.