Year-end Sale % OFF 
Abstract
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.
Highlights
The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) labeled Lys185 in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 °C and pH 7.2, and the labeling inhibited the phosphate transport
The SH reagent eosin 5-maleimide (EMA)1 most significantly interacts with the ADP/ATP carrier; it quickly and labels Cys159 in the second loop facing the matrix of the bovine heart mitochondrial carrier in competition with ADP, showing that the region around Cys159 is a major recognition and binding site of the adenine nucleotides (14 –16)
Effect of FITC on Mitochondrial Proteins—First we examined the labeling of mitochondrial proteins with fluorescein analogs, the SH reagent EMA, and amine/SH-modifier FITC
Summary
Reagents—FITC (isoform I) and EMA were purchased from Molecular Probes (Eugene, OR). Fluorescein, eosin Y, and lysylendopeptidase were from Wako Pure Chemical Industries (Osaka, Japan), erythrosin B was from Fluka (Buchs, Germany), mersalyl was from Aldrich, and hydroxylapatite and AG 1-X8 were from Bio-Rad. E. medium were incubated with 200 M FITC for 10 min at 0 °C in the dark to avoid possible damage of membrane proteins caused by singlet oxygen, which could be generated by fluorescein analogs in the light [14], and the labeling was terminated by 5-fold dilution of samples with the solution used for electrophoresis consisting of 1% SDS, 1% dithiothreitol (DTT), and 25 mM Tris-HCl buffer (pH 6.8). The effects of test compounds on Piuptake by the mitochondria were examined by incubation with a test compound for 10 min at 0 °C and pH 7.2 and determining the Pitransport activity by the addition of a final concentration of 1 mM potassium [32P]phosphate.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
