Related Experiment Video
Updated: May 10, 2026

Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes
María Florencia Leal Denis1, J Jeremías Incicco, María Victoria Espelt
1Department of Biological Chemistry, University of Buenos Aires, Buenos Aires, Argentina.
Background:
The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics).
Methods:
Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry.
Results:
Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers.
Conclusions:
MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers.
General Significance:
Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.
Related Concept Videos
ATP Driven Pumps I: An Overview
There are four main types of ATP-driven pumps - P-type, V-type, F-type, and ABC transporter. All these pumps are of varying complexities and are...
ATP Synthase: Mechanism
ATP Driven Pumps II: P-type Pumps
A typical P-type pump has three cytosolic domains: nucleotide-binding (N), phosphorylation (P), and activator (A) domains. These domains are connected to the membrane-spanning helices by short amino acid segments. ATP hydrolysis and covalent phosphoenzyme intermediate formation are crucial parts of the catalytic cycle. At the highly...
The ADP/ATP Carrier Protein
ATP Synthase: Structure
Primary Active Transport

