Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport.
The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.
Majima
E
E
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
Ishida
M
M
Miki
S
S
Shinohara
Y
Y
Terada
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding; D011487:Protein Conformation; D020539:Sequence Analysis, Protein; D013696:Temperature; D013997:Time Factors
D005456:Fluorescent Dyes; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine