Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Since we have demonstrated that ATPase system was sensitive to chlordecone, it was decided to examine the relationship between physiological and biochemical responses to this neurotoxin. Male Sprague-Dawley rats were fed with chlordecone by gastric intubation at 10, 25 and 50 mg/kg/day for three days. Control rats received 0.3 ml of corn oil. Complete body movements (including tremors) were monitored for a period of 12 hr at 24, 48 and 72 hr after treatment by a piezoelectric crystal attached to the bottom of a plastic rodent cage. The output of the crystal was recorded by a Grass model of EEG machine and magnetic tape. For biochemical study chlordecone treated rats were killed, the brain synaptosomes were prepared and Na+-K+ ATPase, oligomycin-sensitive and insensitive Mg2+ ATPases were determined. Rats receiving chlordecone showed an increased tremor activity which was significant and dose- dependent with a correlation coefficient of the regression line of 0.96. The onset of tremors was evident as early as 2 hr in 50 mg/kg dosed rats. Behavioral abnormalities include startling response to external stimuli like sound, etc. The brain synaptosomal Na+-K+ and oligomycin-sensitive Mg2+ ATPases were significantly decreased in chlordecone treated rats as compared to controls and the decrease was dose-dependent. A linear relationship was observed between the decreases in ATPase activities and physiological (tremor) activity with an r value of 0.96. These results suggest that the inhibition of ATPase system in brain may be related to the production of the neurotoxic symptoms.
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PMID:Acute chlordecone toxicity in rats: a relationship between tremor and ATPase activities. 617 57

The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluorescence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin antibodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin antibodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.
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PMID:Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro. 762 16

Phospholipase A2 [EC 3.1.1.4] treatment of pig kidney Na+,K(+)-ATPase [EC 3.6.1.3] labeled with fluorescence probes at the alpha-chain reduced the extent of the fluorescence intensity change of an N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) probe at Cys-964 to below one-third of the control level accompanying the accumulation of phosphoenzymes. However, it only induced a slight decrease in that of a fluorescence isothiocyanate (FITC) probe at Lys-501 with a large decrease in the rate of change. The addition of phosphatidylserine (PS) or phosphatidylinositol (PI) to the phospholipase-treated BIPM-FITC-labeled enzyme increased the rate of the FITC fluorescence change. Phospholipase treatment of the BIPM-enzyme greatly reduced the Na+,K(+)-ATPase activity. The addition of PS or PI to the treated enzyme induced reactivation. These data and others suggest that Cys-964 and Glu-953 (Rb+ protectable dicyclohexyl carbodiimide binding site) are located in the vicinity of the surface area of the enzyme where hydrocarbon chains of phospholipids are present, and conserved H-bonding amino acids, Thr-955 and Ser-962, are located rather near the center of a domain forming a cation binding route or cage with other hydrophobic transmembrane segments. These data may indicate that the interaction between the BIPM probe and the hydrocarbon chains of phospholipids changes in such a way as to sense the change in the binding state of various ligands accompanying the sequential appearance of reaction intermediates of the enzyme.
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PMID:Different susceptibility to phospholipase A2 treatment of the fluorescence intensity changes in the vicinity of Cys-964 and Lys-501 in the alpha-chain of probe-labeled Na+,K(+)-ATPase. 805 57

An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 micron/s compared to 3-4 micron/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.
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PMID:Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin. 860 51

Free GroEL binds denatured proteins very tightly: it retards the folding of barnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight-binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES.nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES.nucleotide complex. Fast-folding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL.denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL.GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination of folding cage and an annealing machine.
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PMID:Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and -pulsed folding and annealing cage. 863 99

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 A of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts. These results demonstrate that Cys113, Cys172, and Cys422 are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.
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PMID:Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase. 879 5

Muscle mass, distribution of fiber types, fiber cross-sectional areas (CSA) and selected enzyme activities were determined in rats hindlimb-suspended free of immobilization (Susp-Free), suspended with the ankle dorsiflexed (Susp-DF, soleus stretched) or plantarflexed (Susp-PF, soleus shortened) for 10 days and compared to cage-control (Con) rats. Reduction of muscle weight associated with suspension was prevented in Susp-DF rats. The mean CSAs of slow fibers were Con = Susp-DF > Susp > PF > Susp-Free and of fast and intermediate fiber tended to be Susp-DF > Con > Susp-PF = Susp-Free. Mean activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD) and myofibrillar adenosine triphosphatase (mATPase) in slow and fast fibers were similar in Con and Susp-Free rats. Mean SDH activity in slow fibers was higher in Susp-DF and Susp-PF than in Con and Susp-Free. No significant differences in SDH activities of fast fibers were observed among groups. GPD activity was higher in slow fibers of Susp-DF and Susp-PF compared to Con. The mATPase activity was higher in slow fibers of Susp-DF compared to Con and Susp-Free rats and lower in fast fibers of Susp-DF compared to Con rats. Thus, when compared to control, the patterns of adaptation were more similar in the Susp-DF and Susp-PF than in the Susp-Free. Although these results are consistent with previous studies demonstrating that the load placed on a muscle can affect protein metabolism, the direction and magnitude of the adaptive responses observed in the present study were closely associated with the chronically imposed changes in muscle length, i.e. fixed at either a shortened or a lengthened position.
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PMID:Effects of muscle length on the response to unloading. 957 59

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.
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PMID:On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding. 1038 59

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.
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PMID:Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation. 1457 49

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.
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PMID:GroEL mediates protein folding with a two successive timer mechanism. 1514 92


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