High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins

Peter Barr-Gillespie, A. J. Hudspeth

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechanoelectrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect 3,7]decan}-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.

Original languageEnglish (US)
Pages (from-to)625-640
Number of pages16
JournalJournal of Cell Biology
Volume112
Issue number4
StatePublished - Feb 1991
Externally publishedYes

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Rana catesbeiana
Proteins
Sepharose
Auditory Hair Cells
Vestibular Hair Cells
Gels
Stereocilia
Calbindins
Membranes
Glycoside Hydrolases
Nylons
Calmodulin
Tubulin
Biotin
Adhesives
Glass
Actins
Polyacrylamide Gel Electrophoresis
Peptide Hydrolases
Phosphates

ASJC Scopus subject areas

  • Cell Biology

Cite this

High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. / Barr-Gillespie, Peter; Hudspeth, A. J.

In: Journal of Cell Biology, Vol. 112, No. 4, 02.1991, p. 625-640.

Research output: Contribution to journalArticle

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