title: Heme as an optical probe for studying the interactions between calmodulin and the Ca(2+)-ATPase of the human erythrocyte membrane.
abstract: The heme group was used as an optical probe to study the interactions between calmodulin and its targets: the peptide melittin and the enzyme Ca(2+)-ATPase. As already reported, melittin when present in Tris buffer binds hemin-CN which quenches the tryptophan fluorescence. Addition of calmodulin restores the fluorescence significantly accompanied by a blue shift. We show here that the recovery of fluorescence is very slow and takes about 120 min to become constant. In a hydrophobic buffer, the fluorescence spectrum of melittin is already shifted with a peak at 335 nm and intensity almost 2-fold relative to a similar concentration of melittin in Tris buffer. The quenching of tryptophan fluorescence is lesser in this buffer and further addition of calmodulin fails to restore the fluorescence. This indicates the absence of binding of calmodulin to melittin in hydrophobic conditions. Under similar conditions of hydrophobicity, hemin-CN quenches about 35% of the tryptophan fluorescence of the Ca(2+)-ATPase. The subsequent addition of calmodulin restores about half of the quenched fluorescence. The interaction of calmodulin with the Ca(2+)-ATPase even under hydrophobic conditions suggests its high specificity for the enzyme which may be expected for a physiological target.
authors.affiliation: INSERM Unité 299, Hôpital de BicEtre, Le Kremlin BicEtre, France.
authors.forename: A
authors.initials: A
authors.lastname: Zaidi
authors.forename: E
authors.initials: E
authors.lastname: Leclerc-L'Hostis
authors.forename: M C
authors.initials: MC
authors.lastname: Marden
authors.forename: C
authors.initials: C
authors.lastname: Poyart
authors.forename: L
authors.initials: L
authors.lastname: Leclerc
mesh_terms: D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D006418:Heme; D006801:Humans; D008555:Melitten / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D008555:Melitten; D006418:Heme; D000252:Calcium-Transporting ATPases