A MONOCLONAL ANTIBODY (PL/IM 430) THAT BLOCKS THE ACTIVE TRANSL0CATI0N OF Ca²⁺ INTO HUMAN PLATELET INTRACELLULAR MEMBRANE (ER) VESICLES

In earlier studies [1] we identified a number of important biological properties associated with highly purified human platelet intracellular membrane (ER), isolated by continuous flow electrophoresis. These included a high affinity Ca²⁺Mg²⁺ ATPase and protein phosghorylation both of which are involved inthe active uptake of Ca into ER vesicles. The stored Ca²⁺ could be released with inositol(1,4,5)trisphosphate, (IP ), (approx. 50% release in 30 s 1/2 max. for release - 0.25³ μM IP₃,) [2]. To probe the structure-function relationship of proteins in these ER vesicles, a panel of monoclonal antibodies (Mabs) has been raised, using the ER membrane preparation as immunogen. Four of these Mabs recognise a single 100 kDa polypeptide by immunoblotting. This protein is present in platelet membranes and can also be identified in cultured human monocyte, macrophage and endothelial cell lines. None of the M^bs showed any significant effect upon the ER membrane Ca²⁺ Mg²⁺ ATPase activity but one, PL/IM 430 (of IgGl subclass), inhibited the Ca²⁺sequestration by the vesicles significantly (approx. 70% inhibition at 10 μM IgG). This inhibition was independent of the ATP concentration over a range2of 0-2 mM ATP, but was2dose-dependent for external free Ca 2between 30-300 nM Ca²⁺, giving maximum inhibition at 300 nM Ca with 10 pM IgG₂₊ Binding of the antibody substantially lowers the Vmax for Ca₂₊for Ca²⁺ uptake but is without effect upon the Km. PL/IM 430 therefore appears to recognise a 100 kDa polypeptide closely involved with Ca²⁺ trnslocation but at a site which i, s without effect upon the Ca²⁺Mg⁻ ATPase associated with the Ca pump.

We are grateful to the Wellcome Trust and the British Heart Foundation for financial support for these studies.

[1] Hack, N., Croset, M. and Crawford, N. (1986) Biochem. J. 233, 661-668.

[2] Authi, K. S. and Crawford, N. (1985) Biochem. J. Z3O, 247-253