| dc.description | A 49 kDa protein, previously proposed to cross-link microtubules (Campbell et al., 1989), was purified to apparent homogeneity from cell-free extracts of the brine shrimp, Artemia. When incubated with tubulin under assembly conditions, the purified 49 kDa protein cross-linked the resulting microtubules. The 49 kDa protein was moderately resistant to heat, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. The 49 kDa protein cross-links microtubules in a nucleotide-dependent manner. Efficient removal of the 49 kDa protein from microtubules assembled in cell-free extracts of Artemia occurred with GTP and analogues of ATP, nonhydrolyzable or otherwise, but not with ATP itself. The latter nucleotide had a greater impact on cross-linking when microtubules were assembled from purified tubulin. The 49 kDa protein possessed a low level of nucleotidase activity, preferring either ATP or GTP as substrates. Unlike kinesin and dynein, the enzymatic activity of the 49 kDa protein was not stimulated by microtubules. Immunofluorescent staining of Artemia larvae by affinity purified antibodies localized the 49 kDa protein to mitotic spindles, midbodies and setal cells, all regions containing organized microtubules. Regions enriched in microfilaments were not stained with antibody to the 49 kDa protein, nor were microfilaments crosslinked by this protein in vitro. The 49 kDa protein, found in relatively constant amounts at all developmental stages examined, consisted of 4-5 isoforms with isoelectric points in the pH range 6.0-6.2. Isoform composition of the 49 kDa protein may be generated in part by phosphorylation, a posttranslational modification. The observations support the proposal that a novel family of proteins with the ability to modulate microtubule organization in a nucleotide dependent manner exists in Artemia. | en_US |
