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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

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
› Author Affiliations
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.

 

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