Catalytic and regulatory sites in CF1

M.J. Iraburu
M.J. López-Zabalza
E. Santiago
66

Abstract

The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.

Keywords:
Adenosine Diphosphate/pharmacology, Adenosine Triphosphate/pharmacology, Allosteric Site, Anions/pharmacology, Binding Sites, Calcium-Transporting ATPases/chemistry/metabolism, Catalysis, Chloroplasts/enzymology, Enzyme Activation/drug effects, Fluorescein-5-isothiocyanate/pharmacology, Kinetics, Plant Proteins/chemistry/metabolism, Proton-Translocating ATPases/chemistry/metabolism, Trypsin/pharmacology, CF1, Regulatory site, Catalytic site, FITC

Authors

M.J. Iraburu
M.J. López-Zabalza
E. Santiago


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