Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of ¹⁴C ADP was studied with Millipore filtration in a Ca²⁺ and Mg²⁺ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 10⁶ M^-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M^-1. The binding according to the high affinity system showed little temperature dependency (Q₁₀ = 1,10). The pH optimum was at 7,3. Ca²⁺ ions were an absolute requirement for binding.

Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg²⁺ dependent and is inhibited by AMP, in contrast to ADP binding. The Q₁₀ was 1,44.

ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.