title: Evidence for N-Terminal Myristoylation of Tetrahymena Arginine Kinase Using Peptide Mass Fingerprinting Analysis.
abstract: In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi, 780-8520, Japan.
authors.forename: Shou
authors.initials: S
authors.lastname: Motomura
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi, 780-8520, Japan. suzuki@kochi-u.ac.jp.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D001122:Arginine Kinase / Q000737:chemistry*; D019814:Myristic Acid / Q000032:analysis*; D010455:Peptides / Q000737:chemistry; D013769:Tetrahymena pyriformis / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*
chemical_list: D010455:Peptides; D019814:Myristic Acid; D001122:Arginine Kinase
keywords: Arginine kinase; Myristoylation; Peptide mass fingerprinting; Subcellular localization; Tetrahymena pyriformis