Human platelet dense granules: improved isolation and preliminary characterization of [3H]-serotonin uptake and tetrabenazine-displaceable [3H]-ketanserin binding.
An improved method for the isolation of human platelet dense granules was developed. A good yield (45%) of highly enriched (69-fold, based on serotonin content) dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of Km and Vmax values for [3H]-serotonin uptake, as well as the first determination of Kd and Bmax values for tetrabenazine-displaceable [3H]-ketanserin binding, in the human platelet dense granule. The rates and affinities (Vmax 1.45 nmol/mg/min, Km 0.93 uM) of [3H]-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable [3H]-ketanserin binding was observed with a Kd (9.4 nM) similar to, and a Bmax (5.4 pmol/mg) approximately 10-fold lower than, that previously seen in bovine chromaffin granules.
Chatterjee
D
D
Child Study Center, Yale University School of Medicine, New Haven, CT 06511.
Anderson
G M
GM
Chakraborty
M
M
Cohen
D J
DJ
D000135:Acid Phosphatase / Q000032:analysis; D001667:Binding, Competitive; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005165:Factor V / Q000032:analysis; D005952:Glucose-6-Phosphatase / Q000032:analysis; D006801:Humans; D007650:Ketanserin / Q000378:metabolism*; D012701:Serotonin / Q000032:analysis / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013010:Sonication; D013747:Tetrabenazine / Q000378:metabolism; D001620:beta-Thromboglobulin / Q000032:analysis
D001798:Blood Proteins; D001620:beta-Thromboglobulin; D012701:Serotonin; D005165:Factor V; D007650:Ketanserin; D000135:Acid Phosphatase; D005952:Glucose-6-Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D013747:Tetrabenazine