CoChat
Sign Up
Cited by 88Microtubule and mitosis dynamicsProtein Structure and DynamicsEnzyme Structure and Function

Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli.

Gilbert, Susan P., Johnson, Kenneth A.·1993·Biochemistry
DOI 10.1021/bi00068a028
Open this paper in CoChat — free account

Abstract

The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat. Active-site measurements using [alpha-32P]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Integrity signals

Registry

Crossref · 10.1021/bi00068a028

Cites

38 of 38 references verified0 citation errors · 0 not checkable

Statistics

Recomputing p-values needs the paper’s full text.

Claims the paper makes

This paper’s full text isn’t openly available, so its claims and self-check weren’t run. The registry and citation checks still apply.

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The in vitro expressed NH2-terminal motor domain of HC has a higher ATPase activity than the native protein.

    “Similarly, the in vitro expressed NH2-terminal motor domain of HC has a higher ATPase activity than the native protein (Gilbert and Johnson, 1993; Stewart et al., 1993; Huang and Hackney, 1994).”

    From Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules (1998) · cites this paper as Gilbert and Johnson, 1993

  • Inhibition of motor activity by the HC tail is in agreement with previous reports that the motor domain alone has higher MT-stimulated ATPase activity than intact kinesin.

    “Inhibition of motor activity by the HC tail is in agreement with previous reports that the motor domain alone has higher MT-stimulated ATPase activity than intact kinesin (Kuznetsov et al., 1989; Gilbert and Johnson, 1993; Stewart et al., 1993; Huang and Hackney, 1994; discussed in Hackney, 1996).”

    From Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules (1998) · cites this paper as Gilbert and Johnson, 1993

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Gilbert, S. P., & Johnson, K. A.
Affiliations
Pennsylvania State University (US)
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
1993-05-04
Volume
32
Issue
17
Pages
4677-4684
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi00068a028
OpenAlex
W2073764400
Cited by
88
References
38
Citations by year
2023: 3 · 2022: 2 · 2021: 3 · 2020: 2 · 2019: 3 · 2018: 1 · 2017: 1 · 2016: 3 · 2015: 1 · 2014: 4 · 2013: 3 · 2012: 3
FWCI
3.28
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institute of General Medical Sciences (R01GM026726, GM26726)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
Keywords
kinesin motor domain, recombinant protein expression, Escherichia coli expression, ATPase activity, microtubule binding, ADP binding, GTP hydrolysis, ATP binding
MeSH terms
Adenosine Diphosphate, Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Binding Sites, Catalysis, Drosophila melanogaster, Enzyme Activation, Escherichia coli, Guanosine Triphosphate, Kinetics, Microtubules, Plasmids, Recombinant Proteins, Transformation, Bacterial, Gene Expression, Kinesins, GTP Phosphohydrolases

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review