Yuko Kurahashi

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 μmol/min/mg of protein at 37°C. As assayed with 14C-labeled substrates, the apparent K value for anandamide was 60 μM, 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 μmol/min/mg of protein at 37°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.

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.

Panaxynol, a polyacetylene compound isolated from oriental medicines, inhibits mammalian lipoxygenases.

Panaxynol is a polyacetylene compound isolated from commonly used oriental medicines, and its effects on various cyclooxygenases and lipoxygenases were investigated. The compound had only a marginal effect on cyclooxygenase activities (IC50 values >> 100 microM), but inhibited lipoxygenases; 5-lipoxygenase (IC50, 2 microM), two isoforms of 12-lipoxygenase (leukocyte-type, 1 microM; platelet-type, 67 microM) and 15-lipoxygenase (4 microM). Thus, panaxynol inhibited leukocyte-type 12-lipoxygenase much more effectively than platelet-type 12-lipoxygenase. Falcarindiol, an analogue of panaxynol, inhibited these lipoxygenases with higher IC50 values than panaxynol. These compounds could provide a clue to develop a selective inhibitor of one isoform of 12-lipoxygenase.

ANANDAMIDE AMIDOHYDROLASE OF PORCINE BRAIN : CDNA CLONING, FUNCTIONAL EXPRESSION AND SITE-DIRECTED MUTAGENESIS

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.

Brain and Peripheral Anandamide Amidohydrolase and Its Inhibition by Synthetic Arachidonate Analogues

Anandamide (arachidonoyl-ethanolamide, AnNH) was the first brain endogenous substance to be proposed as a physiological agonist at central cannabinoid receptors1. Apart from its cannabimimetic properties (recently reviewed in ref. 2), a role as a neuronal chemical signal for AnNH was suggested also by the finding of molecular mechanisms for its inactivation and Ca2+-dependent biosynthesis by rat central neurons3. The degradation of AnNH by neurons involves the enzymatic hydrolysis of its amide bond with formation of arachidonic acid (AA) and ethanolamine. The enzyme catalyzing this hydrolysis, originally named ‘anandamide amidohydrolase’, was characterized from rat4, 5 and porcine6 brain membranes and was found in high levels also in the liver4. The enzyme was inhibited by typical serine and cysteine protease inhibitors, and displayed a peculiar pH dependency profile with optimal activity at 8.5<pH<10.0. The interest towards AnNH amidohydrolase was increased further by the suggestion2, 8 that the same enzyme may also catalyze the hydrolysis of other bioactive acylethanolamides, such as the anti-inflammatory palmitoylethanolamide (PEA)7, and the primary fatty acid amide, cis-9-octadecenoamide (oleamide), a novel sleep-inducing factor in mammals9. The recent cloning and expression of rat liver ‘oleamide hydrolase’ cDNA10 confirmed that the same enzyme can hydrolyze both oleamide and, at a higher rate, AnNH8, leading to propose for this enzyme the name ‘fatty acid amide hydrolase’ (FAAH)8, 10. In this paper we report the preliminary characterization of FAAH-like enzymatic activities from a blood cell type, the rat basophilic leukemia (RBL-1) cell line, and from other peripheral mammalian and invertebrate cells, and describe the effect of five synthetic AA-derived inhibitors on these enzymes as well as on the previously described amidohydrolases from mouse neuroblastoma (N18TG2) cells8 and porcine brain6.

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.

Catalytic properties of purified recombinant anandamide amidohydrolase.

The major degradative pathway of anandamide, an endogenous ligand for cannabinoid receptors, is its enzymatic hydrolysis to arachidonic acid and ethanolamine.1The enzyme responsible for this reaction has been referred to as anandamide amidohydrolase23or fatty acid amide hydrolase.4‘N-Acylethanolamine amidohydrolase’ reported much earlier by Schmid and co-workers5is probably identical to this enzyme.

Anandamide Amidohydrolase from Porcine Brain

Ethanolamide of arachidonic acid was isolated from porcine brain as an endogenous ligand for cannabinoid receptors, and referred to as anandamide.1 In consideration of various biological activities of anandamide,2 it is very important to elucidate how the production and degradation of this new compound are regulated by enzymes within the cells. The enzyme activity hydrolyzing anandamide to free arachidonic acid and ethanolamine has been considered physiologically important since anandamide loses its biological activity at this step (Fig. 1). Several research groups have reported the anandamide hydrolase activity in the particulate fractions of the brain and other tissues of various animal species.3–5 The specific enzyme activity in these crude preparations was in a range from 0.3 to 9 nmol/min/mg protein. It has also been reported that anandamide is synthesized by the enzymatic condensation of arachidonic acid and ethanolamine. The anandamide synthase activity was found in the brain with a specific activity of 2–3 nmol/min/mg protein.5,6,7 As reported recently, we found both the hydrolase and synthase activities in the microsome fraction of porcine brain,8 from which anandamide was first isolated by Devane and others.1 In this paper we will discuss the partially purified anandamide hydrolase of porcine brain with special reference to its identity with anandamide synthase. We will also describe the inactivation of anandamide by the catalysis of lipoxygenase enzymes.9