A partially purified putative iron P type-ATPase mediates Fe3+-transport into proteoliposome
Section snippets
Materials
All biochemical reagents were from Sigma. 55FeCl3 (2.6 μCi/μL) and [γ-32P] ATP (2 μCi/μL) were from Amersham (Buckinghamshire, UK). BioBeads were from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA) and microfibre glass membranes (GF/C) were from Whatman (Whatman, Inc).
Isolation of nuclei and partial purification of the putative Fe3+-ATPase by nondenaturing-polyacrylamide gel electrophoresis
Rat hepatocytes nuclei were isolated and incubated in standard reaction (SR) medium containing 25 mM Hepes, pH 7.0, 125 mM KCl, 4 mM MgCl2 as previously described [15]. Nondenaturing polyacrylamide gel electrophoresis
ATPase activity, ATP and iron binding under nondenaturing conditions
Fig. 1 shows a nondenaturing-PAGE of nuclear protein extract solubilized with 1 mg/mL of nonionic detergent C12E8, known to preserve sarcoplasmic reticulum Ca2+-ATPase activity [28]. The first step in tracking solubilized Fe3+-ATPase was done by identification of protein band(s) in nondenaturing-PAGE which exhibited ATP hydrolysis. This was observed by formation of white calcium phosphate precipitates after incubation in SR medium, supplemented with 1 mg/mL C12E8, and other additions described in
Discussion
We found that the nonionic detergent C12E8 solubilizes and preserves the activity of nuclear ATPases in agreement with what has been shown for sarcoplasmic reticulum Ca2+-ATPase activity [28]. Solubilized proteins were able to catalyze ATP hydrolysis, as observed by formation of a white calcium phosphate precipitate at the active bands, upon incubation of the gel slab with the substrate. Under these conditions, phosphohydrolytic activity gave rise to the appearance of two major calcium
Acknowledgments
We are thankful to Dr. Hernan Chaimovich and Dr. Yolanda Cuccovia (Biochemistry Department, Institute of Chemistry/University of Sao Paulo, Brazil) for kindly supplying phosphatidylcholine lipids and helping with protocols. We are also thankful to Dr. Nilson Zanchin (National Laboratory of Synchrotron Light, CEBIME, Brazil), Dr. Helotonio Carvalho (Microbiology Department, University of Sao Paulo, Brazil) and Dr. Jeane M. de Freitas (Nutritional Sciences and Toxicology Department, University of
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