Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide

George S. Bloom, Mark Wagner, K. Kevin Pfister, Scott T. Brady

Research output: Contribution to journalArticle

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Abstract

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5′-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124000 (124K) and 64000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 ± 0.21) × 10-7 cm2 s-1 and a sedimentation coefficient of (9.56 ± 0.34) × 10-13 s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379 000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated microtubule-activated ATPase comprising two subunits each of 124000 and 64000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.

Original languageEnglish (US)
Pages (from-to)3409-3416
Number of pages8
JournalBiochemistry
Volume27
Issue number9
StatePublished - 1988
Externally publishedYes

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Kinesin
Brain
Physical properties
Adenosine Triphosphate
Adenylyl Imidodiphosphate
Microtubules
Peptides
Sucrose
Ultracentrifugation
Gel Chromatography
Adenosine Triphosphatases
Gels
Chromatography
Sedimentation
Sodium Dodecyl Sulfate
Stoichiometry
Disulfides
Cytosol
Labeling
Molecular Weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. / Bloom, George S.; Wagner, Mark; Pfister, K. Kevin; Brady, Scott T.

In: Biochemistry, Vol. 27, No. 9, 1988, p. 3409-3416.

Research output: Contribution to journalArticle

Bloom, George S. ; Wagner, Mark ; Pfister, K. Kevin ; Brady, Scott T. / Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. In: Biochemistry. 1988 ; Vol. 27, No. 9. pp. 3409-3416.
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