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 microM 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 -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 P3(1)21 space group. The unit cell dimensions were a = b = 5.89 nm and c = 14.42 nm.
Maatman
R G
RG
Department of Biochemistry, University of Nijmegen, The Netherlands.
Degano
M
M
Van Moerkerk
H T
HT
Van Marrewijk
W J
WJ
Van der Horst
D J
DJ
Sacchettini
J C
JC
Veerkamp
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003124:Colorimetry; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D006110:Grasshoppers; D006801:Humans; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry*; D009363:Neoplasm Proteins; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins