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Biophys J. 1995 Apr;68(4 Suppl):158S-160S; discussion 160S-162S.

Purification of kinesin-related protein complexes from eggs and embryos.

Author information

1
Section of Molecular and Cellular Biology, University of California at Davis 95616, USA.

Abstract

We have developed a biochemical screen for the identification of kinesin-related proteins (KRPs) in their natural host cells and the subsequent purification of these KRPs as native, functional multimeric complexes. The screen involves immunoblotting with pan-kinesin peptide antibodies that recognize several presumptive KRPs in cytosolic extracts; the antibodies have been used so far to monitor the purification of two bona fide kinesin-related motor protein complexes. These two KRPs were purified via AMPPNP-induced microtubule affinity binding, ATP-induced elution from AMPPNP microtubules, gel filtration fractionation, and sucrose density gradient centrifugation. KRP(85/95) from sea urchin (Strongylocentrotus purpuratus) eggs behaves as a heterotrimeric complex of 85-, 95-, and 115-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.4 micron/s. KRP(130) from fruitfly (Drosophila melanogaster) embryos behaves as a homotetrameric complex of four 130-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.04 micron/s. To our knowledge, KRP(85/95) and KRP(130) are the only KRPs to have been purified from native tissue as functional multimeric motor complexes.

PMID:
7787059
PMCID:
PMC1281901
[Indexed for MEDLINE]
Free PMC Article

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