PRIMARY STRUCTURE AND BINDING CHARACTERISTICS OF LOCUST AND HUMAN MUSCLE FATTY-ACID-BINDING PROTEINS

The conservation between muscle fatty-acid-binding proteins (M-FABP) o f Locusta migratoria flight muscle and human skeletal muscle was inves tigated. The locust M-FABP cDNA (632 bp) was isolated by 5' and 3' rap id 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 molecul ar mass of 14935 Da and isoelectric point 6.1. The locust M-FABP was e xpressed in Escherichia coli, purified by (NH4)(2)SO4 precipitation, a nion-exchange and gel-filtration chromatographies and compared with th e recombinant human M-FABP with respect to immunological and binding p roperties. In spite of the high sequence similarity, the proteins did not show immunological cross-reactivity. The binding parameters of loc ust M-FABP were analyzed with radiolabeled oleic acid by the Lipidex a ssay and titration microcalorimetry. Both methods revealed a K-d for o leic acid of 0.5 mu M and a binding stoichiometry of 1 mol fatty acid/ mol FABP. The Delta H, Delta G and Delta S for oleic acid binding were -146 kJ . mol(-1) and -36J . mol(-1) and -369 J . mol(-1) . K-1 respe ctively. All the information obtained from binding, fluorescence and d isplacement studies indicated that locust M-FABP has binding character istics similar to human M-FABP. Finally the recombinant locust M-FABP was crystallized with and without oleic acid. All crystals were trigon al in the P3(1)21 space group. The unit cell dimensions were a = b = 5 .89 nm and c = 14.42 nm.