Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters.
So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).
Böhm
K J
KJ
Institute of Molecular Biotechnology, Beutenbergstrasse 11, Jena, D-07745, Germany. kboehm@ibm-jena.de
Stracke
R
R
Unger
E
E
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000502:physiology; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000502:physiology; D000818:Animals; D006863:Hydrogen-Ion Concentration; D016547:Kinesins / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000502:physiology; D018715:Microscopy, Video; D008870:Microtubules / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000502:physiology*; D009068:Movement / Q000502:physiology*; D009994:Osmolar Concentration; D011487:Protein Conformation; D013552:Swine; D013696:Temperature
D020409:Molecular Motor Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins; D008274:Magnesium