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Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly.

ABSTRACT: The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.

SUBMITTER: Islam K

PROVIDER: S-EPMC1152935 | biostudies-other | 1985 Dec

REPOSITORIES: biostudies-other

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Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.

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Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase.

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SwoHp, a nucleoside diphosphate kinase, is essential in Aspergillus nidulans.

Project description:The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had approximately 20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.

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Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

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Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages.

Project description:Leishmania amazonensis was found to release nucleoside diphosphate kinase (NdK)-a stable enzyme capable of decreasing extracellular ATP. The release of this enzyme from Leishmania results in its progressive accumulation extracellularly as they replicate, peaking at the stationary phase in vitro. The released NdK is immunoprecipitable and constitutes approximately 40% of its total activities and proteins. The retention of a known cytosolic protein by wild type cells and a fluorescent protein by DsRed transfectants at stationary phase, which release NdK, indicates that this is a spontaneous event, independent of inadvertent cytolysis. Recombinant products of Leishmania NdK prepared were enzymatically and immunologically active. Both recombinant and native Leishmania NdK utilized ATP to produce expected nucleoside triphosphates in the presence of nucleoside diphosphates in excess. Both native and recombinant Leishmania NdK were also found to prevent ATP-induced cytolysis of J774 macrophages in vitro, as determined by assays for lactate dehydrogenase release from these cells and for their mitochondrial membrane potential changes. The results obtained thus suggest that Leishmania NdK not only serves its normal house-keeping and other important functions true to all cells, but also prevents ATP-mediated lysis of macrophages, thereby preserving the integrity of the host cells to the benefit of the parasite.

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