Cyclic nucleotide dependent ATPase activity in inside out vesicles from human erythrocytes

Abstract

The transport of cyclic nucleotides out of the cell is energy requiring, dependent on ATP- hydrolysis. The ability to stimulate this ATPase is a hallmark of substrates for ABC-transporters e.g. ABCC5, ABCC4. It is believed that ABCC5 transporter has selective high affinity for cGMP. Previous studies support the idea that ABCC5 contribute to cGMP transport by human erythrocytes. Human erythrocyte membrane possesses cGMP transport system that utilizes ATP for its activity. Present study was conducted to measure, “Cyclic nucleotide dependent ATPase activity in inside out vesicles from human erythrocytes” by an In-house assay method and to compare it with commercially available kit to make cost benefit analysis. The main findings were: 1) The inorganic phosphate standard curves were linear for relevant biological concentrations, observed for both in-house and commercial assay. 2) The membrane protein (i.e. IOV) concentrations raised the inorganic phosphate concentrations linearly. 3) It was possible to distinguish cGMP-stimulated activity from basal ATPase activity in hRBC IOV, even though the difference was small. 4) The two phosphate assays (in-house and commercial) had both advantages and disadvantages, none being superior to the other one.