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Site-specific antibodies to the sea urchin $\beta$-dynein heavy chain

Authors
Publisher
Purdue University
Publication Date
Keywords
  • Biology
  • Molecular
Disciplines
  • Biology
  • Chemistry

Abstract

Dynein is a large multiprotein complex of at least nine subunits. It is responsible for the beating movement of cilia and eukaryotic flagella and is believed to participate in mitosis, fast axonal transport, cellular organization and organelle transport. The dynein subunit that has been of particular interest is the heavy chain. It is a Mg-ATPase and a motor protein. Thus, it has three different activities to: (i) hydrolyze MgATP$\sp{2-}$ and convert it into mechanochemical energy to translocate along microtubules, (ii) interact with microtubules in an ATP-sensitive fashion, and (iii) tether a molecular cargo. With electron microscopy, each dynein heavy chain appears as a head-and-tail structure. An analysis of the available dynein heavy chain sequences does not predict where the different functional and structural domains are on the primary sequence. To better map these structures, a number of site-specific antibodies have been produced to different segments of the sea urchin $\beta$-dynein heavy chain. In addition, there are three autosera reactive with dynein heavy chain that map to the C-terminal third of the polypeptide. The site-specific antibodies have been characterized in solid-phase binding assays and immunoblotting analysis. The antibodies bind specifically to their respective antigens and react with the appropriate photolytic fragment(s). They also cross-react with ciliary and cytoplasmic dyneins from other organisms. Immunoprecipitation assays reveal that some of the antibodies bind to native dynein. The effect of these antibodies on the MgATPase activity of dynein was also examined. The antibody directed against a conserved region in the C-terminal third of dynein heavy chain produced a slight inhibition of dynein enzymatic activity. Peptide-antisera raised against sequence motifs adjacent to the first P-loop were used to correlate the functional classes of the sea urchin egg dyneins. The sea urchin egg contains the 20S soluble ciliary dynein precursor and the cytoplasmic HMr-3 dynein. Immunoblotting analysis indicated that the anti-motif A serum reacted with the 20S soluble egg and embryonic ciliary dyneins and the HMr-3 dynein. In contrast, the anti-motif B serum reacted with only the HMr-3 dynein. By indirect immunofluorescence microscopy, the anti-motif A serum stained embryonic cilia while the anti-B serum did not. These results demonstrate that the two egg dyneins have distinct protein sequences and that the motif B dyneins are excluded from cilia. This observation is consistent with the hypothesis that the 20S soluble dynein is a ciliary dynein precursor. ^

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