Purification of kinesin-related protein complexes from eggs and embryos

D. G. Cole, J. M. Scholey, S. Endow, C. Schutt, E. Young, D. Hackney, D. White, S. Block, P. Satir

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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 μm/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 μm/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.

Original languageEnglish (US)
JournalBiophysical Journal
Volume68
Issue number4 SUPPL.
StatePublished - 1995

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Egg Proteins
Kinesin
Embryonic Structures
Microtubules
Proteins
Adenylyl Imidodiphosphate
Strongylocentrotus purpuratus
Density Gradient Centrifugation
Sea Urchins
Antibodies
Drosophila melanogaster
Immunoblotting
Eggs
Gel Chromatography
Sucrose
Adenosine Triphosphate
Peptides

ASJC Scopus subject areas

  • Biophysics

Cite this

Cole, D. G., Scholey, J. M., Endow, S., Schutt, C., Young, E., Hackney, D., ... Satir, P. (1995). Purification of kinesin-related protein complexes from eggs and embryos. Biophysical Journal, 68(4 SUPPL.).

Purification of kinesin-related protein complexes from eggs and embryos. / Cole, D. G.; Scholey, J. M.; Endow, S.; Schutt, C.; Young, E.; Hackney, D.; White, D.; Block, S.; Satir, P.

In: Biophysical Journal, Vol. 68, No. 4 SUPPL., 1995.

Research output: Contribution to journalArticle

Cole, DG, Scholey, JM, Endow, S, Schutt, C, Young, E, Hackney, D, White, D, Block, S & Satir, P 1995, 'Purification of kinesin-related protein complexes from eggs and embryos', Biophysical Journal, vol. 68, no. 4 SUPPL..
Cole DG, Scholey JM, Endow S, Schutt C, Young E, Hackney D et al. Purification of kinesin-related protein complexes from eggs and embryos. Biophysical Journal. 1995;68(4 SUPPL.).
Cole, D. G. ; Scholey, J. M. ; Endow, S. ; Schutt, C. ; Young, E. ; Hackney, D. ; White, D. ; Block, S. ; Satir, P. / Purification of kinesin-related protein complexes from eggs and embryos. In: Biophysical Journal. 1995 ; Vol. 68, No. 4 SUPPL.
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AU - Schutt, C.

AU - Young, E.

AU - Hackney, D.

AU - White, D.

AU - Block, S.

AU - Satir, P.

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