⁷⁵Se-Seleno-Methionine-Actin as a Probe for Determination of Platelet Production Rate

Summary

Synthesis of actin is a reasonably correct representation of the platelet production rate. Measurement of actin production obviates some of the drawbacks encountered in currently available methods. Platelet actin was characterized on polyacrylamide gel electrophoreses using methylated radioactive rabbit actin as a reference. Platelet actin was unambiguously identified by showing that it forms a complex with DNAase-I similar to the complex obtained with pure rabbit actin.

Six days after intravenous injection of ⁷⁵Se-seleno-methionine actin was one of the most highly labelled platelet components. The platelet pellet (one rat per sample) was solubilized with 1% Triton X-100, 0.75 M guanidine-HCl and dialyzed in a medium containing 1% SDS (to eliminate Triton X-100 and guanidine-HCl). A carefully measured aliquot was deposited on a polyacrylamide gel. After electrophoresis in the presence of SDS the radioactivity of the actin band was measured and the ratio of the total actin radioactivity to the injected radioactivity was taken as a measure of platelet production.

The validity of the actin probe was tested with populations of normal animals. The specific radioactivity of actin was proportional to the injected dose of ⁷⁵Se-seleno-methionine up to 1 mCi/ kg animal. A semi-log plot of actin specific radioactivity vs time exhibited a pseudo-first order decrease. Another constituent with a high specific radioactivity (X_M) was excluded as a suitable probe because it was shown to be an adsorbed plasma protein.

Keywords

Platelet production rate - Actin - Actin-DNAase-I complex actin labelling by reductive methylation - Electrophoresis - Platelet kinetics

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