A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.
To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.
Hack
N
N
Department of Biochemistry, Hunterian Institute, Royal College of Surgeons of England, London, U.K.
Wilkinson
J M
JM
Crawford
N
N
D000255:Adenosine Triphosphate / Q000097:blood; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology*; D010070:Oxalates / Q000494:pharmacology; D010455:Peptides / Q000097:blood; D013861:Thiocyanates / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D008565:Membrane Proteins; D010070:Oxalates; D010455:Peptides; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium