Primary structure and binding characteristics of locust and human muscle fatty‐acid‐binding proteins

The conservation between muscle fatty‐acid‐binding proteins (M‐FABP) of Locusta migratoria flight muscle and human skeletal muscle was investigated. The locust M‐FABP cDNA (632 bp) was isolated by 5′ and 3′ rapid amplification of cDNA ends. The identities of the locust and human M‐FABP on the cDNA and protein levels were 54% and 42%, respectively. The predicted amino acid sequence of locust M‐FABP indicated a molecular mass of 14935 Da and isoelectric point 6.1. The locust M‐FABP was expressed in Escherichia coli, purified by (NH4)2SO4 precipitation, anion‐exchange and gel‐filtration chromatographies and compared with the recombinant human M‐FABP with respect to immunological and binding properties. In spite of the high sequence similarity, the proteins did not show immunological cross‐reactivity. The binding parameters of locust M‐FABP were analyzed with radiolabeled oleic acid by the Lipidex assay and titration microcalorimetry. Both methods revealed a Kd for oleic acid of 0.5 μM and a binding stoichiometry of 1 mol fatty acid/mol FABP. The ΔH, ΔG and ΔS for oleic acid binding were −146 kJ · mol−1 and −36 J · mol−1 and −369 J · mol−1· K−1 respectively. All the information obtained from binding, fluorescence and displacement studies indicated that locust M‐FABP has binding characteristics similar to human M‐FABP. Finally the recombinant locust M‐FABP was crystallized with and without oleic acid. All crystals were trigonal in the P3121 space group. The unit cell dimensions were a=b= 5.89 nm and c= 14.42 nm.