The ATP hydrolytic activity of SPF1 ATPase

CORRADI GERARDO RAUL; DE TEZANOS PINTO FELICITAS; GAUTIER MARIEN; ADAMO HUGO PEDRO

Congreso; XXXIX Annual Meeting of Argentinean Biophysical Society SAB 2010, workshop CeBEM, structural biology in Latin America, 3rd Latin American Protein Society Meeting; 2010

The ion specificity of the ion transporting P5-ATPases is not known1. Mutations in these transporters have been linked to hereditary Parkinsonism and Autism spectrum disorder2. We have previously reported the overexpression of the yeast P5-ATPase SPF1p in Saccharomyces cerevisiae3. The recombinant protein had and amino terminal polyhistidine-green fluorescent protein tag, for ease the purification and detection. We found that the purified protein was able to hydrolyze ATP at 0.2 mmol/mg/min at 37 ºC in a reaction media that contained 20 mM Hepes-K (pH 7.2 at 37 ºC ), 25 mM imidazol, 100 mM KCl, 0.5 mM EGTA, 2mM MgCl2, 3 mM ATP, 1 mg of purified protein and 0.016% of C12E10.  When the purified protein was supplemented with 0.1% phosphatidylethanolamine (PE) the activity slightly increased to 0. 3 mmol/mg/min, while it rose to 0.8 mmol/mg/min when supplemented with either phosphatidylcholine  (PC) or a mixture of brain acidic phospholipids (BE). The possibility that the ATPase activity was originated from traces of contaminating proteins was evaluated by measuring the activity of a mutant protein in which the catalytic residue Asp487 was substituted by Asn. The preparation of purified mutant was able to hydrolyze ATP, but the rate was 45% of that of the wild type enzyme. Thus despite the background ATPase due either to the residual activity of the mutant Asp487Asn or because of the presence of contaminating ATPases, these results show that the purified SPF1 protein has the intrinsic ability to hydrolyze ATP and that this activity is optimal when supplemented with PC.