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Thromb Haemost 1978; 39(02): 386-403
DOI: 10.1055/s-0038-1646699
Original Article
Schattauer GmbH Stuttgart

The Subcellular Distribution of [3H]-Colchicine-Binding Activity and Tubulin in Pig Blood Platelets

Authors

  • Alan G Castle

    The Department of Biochemistry, University of Birmingham, P. O. Box 363, Birmingham B15 2TT, England

  • Neville Crawford

    The Department of Biochemistry, University of Birmingham, P. O. Box 363, Birmingham B15 2TT, England
Further Information

Publication History

Received 04 April 1977

Accepted 05 September 1977

Publication Date:
12 July 2018 (online)

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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.