title: Cytochrome c forms complexes and is partly reduced at interaction with GPI-anchored alkaline phosphatase.
abstract: Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, 61137, Brno, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Dadák
authors.forename: Oldrich
authors.initials: O
authors.lastname: Janiczek
authors.forename: Oldrich
authors.initials: O
authors.lastname: Vrána
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
chemical_list: D003574:Cytochrome c Group; D017261:Glycosylphosphatidylinositols; D000469:Alkaline Phosphatase