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mM ATP (mm + atp)
Selected AbstractsStrikingly fast microtubule sliding in bundles formed by Chlamydomonas axonemal dynein,CYTOSKELETON, Issue 6 2010Susumu Aoyama Abstract Chlamydomonas axonemal extracts containing outer-arm dynein bundle microtubules when added in the absence of ATP. The bundles dissociate after addition of ATP (Haimo et al., Proc Natl Acad Sci USA 76:5759,5768, 1979). In the present study, we investigated the ATP-induced bundle dissociation process using caged ATP. Application of ,0.5 mM ATP induced microtubule sliding at ,30 ,m·s,1, which was 1.5 times faster than the microtubule sliding observed in protease-treated axonemes and five times faster than microtubule gliding on glass surfaces coated with outer-arm dynein. Bundles formed by mutant dynein molecules that lack one of the three heavy chains (HCs) displayed similar high-speed intermicrotubule sliding. These results suggest that Chlamydomonas outer-arm dynein molecules, when aligned, can translocate microtubules at high speed and that the high-speed sliding under load-free conditions does not require the complete set of the three HCs. It is likely that each of the three HCs has the ability to produce high-speed sliding, which should be an important property for their cooperation. © 2010 Wiley-Liss, Inc. [source] Regulation of monomeric dynein activity by ATP and ADP concentrationsCYTOSKELETON, Issue 4 2001Katsuyuki Shiroguchi Abstract Axonemal dyneins are force-generating ATPases that produce ciliary and flagellar movement. A dynein has large heavy chain(s) in which there are multiple (4,6) ATP-binding consensus sequences (P-loops) as well as intermediate and light chains, constituting a very large complex. We purified a monomeric form of dynein (dynein- a) that has at least three light chains from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein- a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP (, 20 ,M). Both ATPase activity and translocational velocities in the presence of ADP (, 20 ,M) fit the Michaelis-Menten equation well. However, in the absence of ADP (< 0.1 ,M), neither of the activities followed the Michaelis-Menten-type kinetics, probably due to the effect of two ATP-binding sites. Our results also indicate that dynein- a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of a dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system. Cell Motil. Cytoskeleton 49:189,199, 2001. © 2001 Wiley-Liss, Inc. [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE -INDUCED DEFICIENCY OF METHYL DONORSJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2000R. Bianchi We used cycloleucine (CL) , which prevents methionine conversion to S-adenosyl-methionine (SAMe) by inhibiting ATP-L-methionine-adenosyl-transferase (MAT) , to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35,50%. SAMe p-toluensulphonate (SAMe-SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL-treated animals killed at day 18 after birth, starting from labelled substrates ([3H]-choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL-treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30,40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL. Among proteins, in sciatic nerves of CL-treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe-SD4 treatment counteracted, and in some cases normalized, these changes. In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe-SD4 administration. The financial support of Telethon-Italy (grant No. D 51) is gratefully acknowledged. [source] Roles of the two ClpC ATP binding sites in the regulation of competence and the stress responseMOLECULAR MICROBIOLOGY, Issue 3 2001Kürsad Turgay MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10-fold but was not eliminated. In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC. [source] NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATPBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2003Brigitte Pelzmann The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36,37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP-dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 ,g ml,1 NADH (4,6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF,1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg,1 protein versus control: 35.4±2.57 nmol mg,1 protein, P<0.000005). These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP. British Journal of Pharmacology (2003) 139, 749,754. doi:10.1038/sj.bjp.0705300 [source] Azotobacter vinelandii Metal Storage Protein: "Classical" Inorganic Chemistry Involved in Mo/W Uptake and Release ProcessesCHEMBIOCHEM, Issue 4 2008Jörg Schemberg Dr. Abstract The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12,°C revealed that three different steps can be distinguished for the Mo-release process. A proportion of ,15,% at pH 6.8,7.0, an additional 25,% at pH 7.2,7.5 and ca. 50,% (up to 90,% in total) at pH 7.6,7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30,°C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play,in addition to the pH value,an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110,115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12,°C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate,ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (,110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed.2007, 46, 2408,2413). [source] | |||||||||||||