Summary
The subcellular distribution of the [3H]-colchidne-binding protein, believed to be tubulin, the subunit protein of microtubules,
has been investigated in mammalian blood platelets. Studies on a soluble extract from
pig platelets and two particulate fractions (viz. membrane-rich and granule-rich fractions)
have shown that about 98% of the colchicine-binding activity in a platelet homogenate
is located in the soluble phase. This result is in agreement with poly-acrylamide
gel electrophoresis experiments which show that the soluble fraction contains a substantial
amount of 55,000 MW tubulin, whereas the membrane and granule-rich fractions contain
very little of this component. The [3H]-colchicine-binding activity of the platelet soluble phase is largely precipitated
by 40-50% ammonium sulphate and also by vinblastine sulphate in millimolar concentrations.
Moreover the colchicine-binding protein in the platelet soluble fraction has a sedimentation
coefficient of 5.9 S, is eluted in the void volume of a Sephadex G-100 column, and
binds to DEAE-Sephadex at low ionic strength and is eluted from this ion-exchanger
at an ionic strength of 0.47 M-KC1. In addition, most of the col-chi cine-binding
activity of the platelet soluble phase is associated with protein which will undergo
temperature-dependent polymerization in vitro and which has a molecular weight on SDS-polyacrylamide gels of 55,000. All these
experimental findings suggest that the col-chi cine-binding activity of pig platelet
homogenates is due to the presence of the microtubule protein, tubulin, which is largely
found in the soluble compartment of the cells.
