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Drosophila kinesin: characterization of microtubule motility and ATPase
W M Saxton1, M E Porter, S A Cohn
1Department of Biology, Indiana University, Bloomington 47405.
Abstract:
Preparations of kinesin, a microtubule-based force-producing protein, have been isolated from Drosophila melanogaster embryos by incubation of microtubules with a nonhydrolyzable ATP analogue and gel filtration of proteins released from the microtubules by ATP. These preparations induced MgATP-dependent microtubule gliding in vitro with a Km for MgATP of 44 microM and a Vmax for gliding of 0.9 micron/sec. Samples of Drosophila proteins that were active in motility assays possessed an average ATPase activity in solution of 17 nmol/min per mg that increased to an average of 106 nmol/min per mg in the presence of microtubules. The major polypeptides that copurified with these activities showed relative molecular masses of 115 kDa and 58 kDa. An antiserum raised against the 115-kDa polypeptide also recognized the 110-kDa component of squid kinesin preparations and the 130-kDa component of sea urchin kinesin preparations.
Insights
Researchers isolated kinesin, a microtubule motor protein, from fruit fly embryos. This purified kinesin drives microtubule gliding in vitro, demonstrating its force-producing capabilities.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Kinesin is a crucial microtubule-based motor protein responsible for intracellular transport.
- Understanding kinesin's function requires isolating and characterizing active protein preparations.
Purpose of the Study:
- To isolate and characterize force-producing kinesin from Drosophila melanogaster embryos.
- To assess the in vitro motility and biochemical properties of purified Drosophila kinesin.
Main Methods:
- Kinesin isolation using ATP analogue incubation and gel filtration from Drosophila embryo extracts.
- In vitro microtubule gliding assays to measure motility parameters (Km, Vmax).
- ATPase activity assays in solution and in the presence of microtubules.
- Polypeptide identification via gel electrophoresis and Western blot analysis using antiserum.
Main Results:
- Isolated preparations demonstrated MgATP-dependent microtubule gliding with specific kinetic parameters (Km = 44 μM, Vmax = 0.9 μm/sec).
- ATPase activity increased significantly in the presence of microtubules (17 to 106 nmol/min/mg).
- Major copurifying polypeptides were identified at 115 kDa and 58 kDa; the 115-kDa polypeptide showed cross-reactivity with kinesin from other species.
Conclusions:
- Drosophila melanogaster embryos are a viable source for isolating functional kinesin.
- The purified kinesin exhibits ATP-dependent motor activity and specific biochemical characteristics.
- The 115-kDa polypeptide is likely a component of Drosophila kinesin, with conserved epitopes across species.