Nantly present in pellet fraction further indicating that FAAH may be associated with other intra cellular membrane bound organelles. The small quantities of FAAH in the supernatant after this spin strongly suggest a predominantly membrane associated protein and is further supported PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10572343 by increased yields of HIS-FAAH when detergents such as Triton X-100 are added. Unlike other mammalian FAAHs, Dictyostelium FAAH does not have any predicted transmembrane domain. Similar membrane associated behaviour was reported when humanFAAH was expressed as a recombinant protein lacking a N-terminal transmembrane domain and the protein was predominantly present in membrane fractions [23].Figure 6 Western blotting analysis of distribution of HIS-FAAH in membrane fractions of Dictyostelium. Total cellular protein (L) from AX3FAAH cells were fractionated into 13,000xg membrane and cytosol fractions (P1 and S1 respectively) and 100,000xg membrane and cytosolic fractions (P2 and S2 respectively). Described membrane and cytosolic fractions were separated on 10 SDS-PAGE and subjected to Western blotting using anti-HIS antibody. M represents molecular mass standard in kDa.Discussion Bioinformatics analysis of FAAH amino acid sequence revealed the presence of an amidase signature domain, which is similar to that present in other mammalian FAAH. The amidase signature sequence is conserved among many proteins from the amidase class, 5-Tris(4 which include enzymes hydrolyzing acetamide, acrylamide, nicotinamide, and glutamide [24-27]. FAAH is the only characterized mammalian enzyme belonging to the amidase class and recently the FAAH homolog from Arabidopsis has been characterized and reported to belong to the amidase class. Despite Dictyostelium FAAH’s considerable deviations in sequence identity across full length amino acid sequences when compared to human, porcine, rat and Arabidopsis sequences, Dictyostelium FAAH has retained anandamide hydrolysis function. Recombinant FAAH produced from Dictyostelium and E.coli was capable of hydrolyzing anandamide and other fatty acid substrates arachidonoyl p-nitroaniline and decanoyl p-nitroaniline similar to other characterized FAAHs. Previously, Schmid and co-workers reported N-acylethanolamine amidohydrolase from rat liver which hydrolyzed various N-acylethanolamines [28] but did not test anandamide as a substrate. Later when Cravatt’s group cloned and characterised N-acylethanolamine amidohydrolase cDNA, the enzyme hydrolysed anandamide in addition to other fatty acid amides. These findings indicated (S)-tert-Butyl 6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate that the enzyme may regulate growing family of bioactive fatty acid amides, and the enzyme was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/21715270 renamed as fatty acid amide hydrolase. Kinetic parameters indicate that Dictyostelium FAAH has preferred affinity for longer unsaturated acyl chains and inhibition by PMSF, LY2183240 and MAFP suggest a conserved enzyme mechanism between Dictyostelium and mammalian FAAH [29,30]. These preliminary biochemical and kinetic analyses of Dictyostelium FAAH supports the identification of [GenBank: XM_638290] as a functional homolog of mammalian FAAH. N-acylphosphatidylethanolamines (NAPEs) and its hydrolysed product N-acylethanolamines 2-Amino-3-methoxypyridine (NAEs) have been previously reported in Dictyostelium [31]. Identification of FAAH in Dictyostelium indicates FAAH may be a potential regulator of NAEs produced in Dictyostelium cells. Among many established physiological roles for anandamide in mammalian cells, recently a role in neutrophil chemotaxis was identifi.