Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs

J. M. Scholey, B. Neighbors, J. R. McIntosh, E. D. Salmon

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61 Scopus citations

Abstract

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis, and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a M(r) = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The M(r) = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where >90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATPase co-purified with high M(r) MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5m and hydroxylapatite chromatography. The high M(r) MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (M(r) ~ 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this M(r) ~ 350,000 polypeptide is cytoplasmic dynein.

Original languageEnglish (US)
Pages (from-to)6516-6525
Number of pages10
JournalJournal of Biological Chemistry
Volume259
Issue number10
StatePublished - 1984
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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