Enzyme reaction was initiated by adding 10g of HIS-FAAH or MBP-FAAH enzyme (from 0

Enzyme reaction was initiated by adding 10g of HIS-FAAH or MBP-FAAH enzyme (from 0.2?mg?ml-1 stock, in 20?mM Tris-Cl, pH 9.0, 50?mM NaCl) and incubated at 37C for 30?min and the reaction was analyzed by CE-ES-MS. the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. Conclusions This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from (http://dictybase.org/gene) [GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_638290″,”term_id”:”66819444″,”term_text”:”XM_638290″XM_638290] containing coding sequences for characteristic amidase signature motifs [19] was identified and found to be located on chromosome 2 in the annotated Dictyostelium genome data base. [GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_638290″,”term_id”:”66819444″,”term_text”:”XM_638290″XM_638290] will be referred to as Dictyostelium FAAH as the proteins amino acid sequence analysis and other TPO agonist 1 experimental results confirm its function to be much like mammalian FAAH. The calculated molecular excess weight of Dictyostelium FAAH is usually 70?kDa and domain name architecture analysis (http://www.ncbi.nlm.nih.gov/structure/cdd) reveals the presence of an amidase domain name composed of a characteristic amidase signature (AS) sequence (Physique ?(Figure1).1). The consensus amidase signature sequence has a conserved GSS(G/A/S)G (residues 304 to 308) motif shared among many proteins in the amidase class including glutamyl-t-RNA amidotransferase subunit A of and FAAH from human, porcine, rat, Arabidopsis and Dictyostelium. FAAH from human, porcine and rat are composed of 579 amino acids and FAAH from Dictyostelium and Arabidopsis contain 637 and 607 amino acids, respectively. FAAH full length protein amino acid sequence from Dictyostelium lacks significant identity when compared to FAAH from human (20%), porcine (20%), rat (20%), and Arabidopsis (32%) (Physique ?(Figure1),1), but identity across the amidase signature sequence increased to 40%, 38%, 38%, and 50%, for the human, procine, rat, and Arabidopsis FAAH homologs. The serine residues at 217 and 241 found to be essential for rat FAAH activity [20] were also conserved in AS sequence of Dictyostelium FAAH. Other catalytically important residues Lys142, Ser218 and Arg243 found in rat were also conserved in Dictyostelium. Open in a separate window Physique 1 Comparative alignment of amino acid sequences of Dictyostelium FAAH with mammalian Dll4 and Arabidopsis FAAH. Full length amino acid sequence alignment of human [NCBI:”type”:”entrez-protein”,”attrs”:”text”:”NP_001432″,”term_id”:”166795287″,”term_text”:”NP_001432″NP_001432], porcine [NCBI:”type”:”entrez-protein”,”attrs”:”text”:”NP_999079″,”term_id”:”47522660″,”term_text”:”NP_999079″NP_999079], rat [NCBI:”type”:”entrez-protein”,”attrs”:”text”:”NP_077046″,”term_id”:”13162304″,”term_text”:”NP_077046″NP_077046], Arabidopsis (AT) [NCBI:”type”:”entrez-protein”,”attrs”:”text”:”AAP83139″,”term_id”:”32454719″,”term_text”:”AAP83139″AAP83139] and Dictyostelium (Dicty) [NCBI:”type”:”entrez-protein”,”attrs”:”text”:”XP_643382″,”term_id”:”66819445″,”term_text”:”XP_643382″XP_643382]. The amidase signature (AS) sequence is usually underlined and TPO agonist 1 consists of about 126 amino acids. Asterisks denote identical amino acids and residues essential for FAAH activity in rat is usually indicated by arrow mark. Recombinant enzyme expression and affinity purification of FAAH in Dictyostelium and were not successful, as both N-terminal HIS and C-terminal HIS fusions to FAAH were unstable and only a small amount of the protein was made and this was only found in inclusion bodies. Alternatively, in order to simplify large scale recombinant protein production, FAAH was expressed and purified as a recombinant maltose binding protein (MBP) fusion protein from (Physique ?(Physique2D,2D, E). Recombinant FAAH when expressed as N-terminal MBP fusion protein (MBP-FAAH) in produced a higher yield of soluble recombinant protein. Recombinant FAAH when produced in either Dictyostelium or migrated on SDS-polyacrylamide gels, consistent with no significant post-translation modification. Open in a separate window Physique 2 (A) Coomassie staining of purified HIS-FAAH recombinant protein from Dictyostelium. Dictyostelium cells AX3FAAH expressing HIS-FAAH were lysed and the recombinant protein was bound to Ni-NTA resin. Resin bound protein was eluted using lysis buffer made up of 200?mM Imidazole and the eluate fractions S1, S2, S3, S4, S5 were resolved on 10% SDS-PAGE and Coomassie stained. (B) Western blotting analysis. Fractions analysed in TPO agonist 1 Physique 2A were analysed by Western blotting using anti-HIS antibody. (C) TPO agonist 1 Western blotting analysis. Fractions analysed in Physique 2A/2B were pooled together (P1) and analysed by Western blotting using anti-FAAH polyclonal antibody and the same portion was used in enzyme kinetic assay. (D) Coomassie staining analysis of purified recombinant MBP-FAAH protein from hydrolyzed anandamide to free arachidonic acid and ethanolamine as determined by CE-ES-MS (Physique ?(Physique3A,3A, B, C). Dictyostelium FAAH was also capable of hydrolyzing synthetic (A) CE-ES-MS analysis of control reaction having anandamide alone in the reaction buffer without enzyme was analyzed. Negative ion mode product ion scan.