Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly-competent

AbstractChick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP-induced assembly is not the result of generation of GTP by the co-purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25–35% is assembled into other structural forms in the presence of ATP

Are guanine nucleotide binding proteins a distinct class of regulatory proteins?

AbstractProteins which bind guanine nucleotides are found in a diverse set of key regulatory positions. They are involved in hormone action, visual transduction, protein synthesis and microtubule assembly. In addition to their ability to bind guanine nucleotides these proteins possess several other common features. (i) They all have similar subunit composition, (ii) they can be ADP-ribosylated, (iii) their conformation changes depending on the nucleotide bound. These regulatory G-proteins have close functional homologies. Do they form a general class of regulatory proteins, like the protein kinases? Do they have a common evolutionary ancestry

Induction of anti-actin drug resistance in Tetrahymena

Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 μM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross-resistance to cytochalasin D. However, much higher \u3e30 μM) concentrations of cytochalasin D were required for induction of cross-resistance to latrunculin B. Anti-actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F-actin assembly and stability

Relative potencies of different cytochalasins for the inhibition of phagocytosis in ciliates

Ciliate actins have been reported to exhibit an unusual degree of sequence divergence, within the phylum Ciliophora, and when compared to actins from other organisms. To determine whether these primary structural differences are correlated with pharmacological differences, we investigated the effects of seven cytochalasins on phagocytosis, which has been shown to be actindependent in eukaryotic cells. The relative potencies of cytochalasin inhibition of phagocytosis in Spirostomun ambiguum and Paramecium multimicronucleatum were similar. Dihydrocytochalasin B and cytochalasin A were the most potent of the seven cytochalasins in both ciliates, and strongly inhibited phagocytosis at 20-40 μM. Dihydrocytochalasin B was considerably more potent than either cytochalasins B or D, a result unexpected on the basis of reports utilizing other organisms. However, even at concentrations up to 100 μM, dihydrocytochalasin B did not inhibit the rate of Spirostomum defecation. After long-term treatment of Spirostomum with 50 μM dihydrocytochalasin B, the sensitivity of phagocytosis to the drug was reduced. These results indicate that the relative cytochalasin sensitivities of actin-dependent functions in ciliates differ from those of other organisms, and are consistent with the hypothesis that sequence differences might give rise to substantial differences in the pharmacological properties of ciliate actins