title: ATP protects against FITC labeling of Solanum lycopersicon and Arabidopsis thaliana Ca2+-ATPase ATP binding domains.
abstract: Ca(2+)-ATPases are integral membrane proteins that actively transport Ca(2+) against substantial concentration gradients in eukaryotic cells. This active transport is energized by coupling ion translocation with ATP hydrolysis. In order to better understand this coupling mechanism, we studied the nucleotide specificities of isolated ATP binding domains (ABDs) of Solanum lycopersicon Ca(2+)-ATPase (LCA), a type IIA non-calmodulin regulated P-type pump found in tomato plants that is very similar to mammalian sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), and Arabidopsis Ca(2+)-ATPase, isoform 2 (ACA2), a type IIB calmodulin regulated P-type ATPase found in the endoplasmic reticulum of Arabidopsis cells. We used nucleotide protection against FITC labeling as a measure of binding since both LCA and ACA contained the KGAP(S,V,F)E motif, which has been shown to be modified by fluorescein isothiocyanate (FITC) in P-type pumps from animal cells. We demonstrated that the heterologously expressed GST-tagged ABDs from both LCA and ACA2 were modified by FITC and that ATP protects against this modification. Moreover, GTP was able to reduce, but not eliminate, the level of FITC labeling in both ABD constructs, suggesting that these plant pumps may also bind GTP with low affinity, which is in contrast to mammalian SERCA and PMCA type pumps which do not bind GTP.
authors: School of Biological Sciences, Illinois State University, Normal, IL 61790, USA.
authors: Charitha
authors: C
authors: Galva
authors: Gail K
authors: GK
authors: Virgin
authors: Jeff B
authors: JB
authors: Helms
authors: Craig
authors: C
authors: Gatto
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D032322:Solanum / Q000201:enzymology*
chemical_list: D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate
keywords: ACA2; Ca(2+)-ATPase; FITC; LCA
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