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

R. G H J Maatman, M. Degano, H. T B Van Moerkerk, W. J A Van Marrewijk, D. J. Van Der Horst, J. C. Sacchettini, J. H. Veerkamp

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)801-810
Number of pages10
JournalEuropean Journal of Biochemistry
Volume221
Issue number2
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry

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    Maatman, R. G. H. J., Degano, M., Van Moerkerk, H. T. B., Van Marrewijk, W. J. A., Van Der Horst, D. J., Sacchettini, J. C., & Veerkamp, J. H. (1994). Primary structure and binding characteristics of locust and human muscle fatty-acid-binding proteins. European Journal of Biochemistry, 221(2), 801-810.