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 K(d) 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 MFABP. 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.
|Number of pages||10|
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1994|
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