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)

The aim of the present study was to further subdivide the type II fibers of the human thyroarytenoid and posterior cricoarytenoid muscles by means of a modified myosin ATPase reaction. In order to understand the functioning of these highly strained muscles better, it is important to know the respective percentage of fatigue-resistant type IIA fibers and fatigable type IIB fibers. The material comprised the larynges of seven laryngectomized males aged between 45 and 70 years and four laryngectomized females aged between 39 and 72 years. After having been frozen in nitrogen, 10-microns-thick sections were cut from the laryngeal muscles in a cryostat. The pH-lability of the enzyme that can be utilized in a classical myosin ATPase reaction permits a differentiation between fiber types I, IIA and IIB. Evidently, this is not possible with every human muscle. The fiber types IIA and IIB of the thyroarytenoid and the posterior cricoarytenoid muscles could be clearly distinguished by means of the inhibition reactivation myofibrillar ATPase technique. Using this method, the myosin ATPase enzyme was initially inhibited by hydroxymercuribenzoate and subsequently reactivated by cysteine. Regarding the incidence of type I and IIA fibers, there was a statistically significant difference between the thyroarytenoid and the posterior cricoarytenoid muscles. The type IIA fiber content was statistically significantly higher in the arytenoid muscle than in the posterior cricoarytenoid muscle. The percentage of type IIB fibers was low, not only in the thyroarytenoid muscle and the posterior cricoarytenoid muscle but also in the other laryngeal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fiber differentiation of the human laryngeal muscles using the inhibition reactivation myofibrillar ATPase technique. 141 83

A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.
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PMID:Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing. 141 61

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.
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PMID:Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function. 142 Sep 10

A novel approach for locating sites of interest in a protein complex has been developed using monomaleimidonanogold (MMN). The Escherichia coli F1 ATPase, when prepared without the delta subunit, contains only a single reactive cysteine on one of the three copies of the alpha subunit. This site was reacted with MMN and the gold cluster visualized on the protein complex by cryoelectron microscopy. Additional sites for modification with MMN were added by introducing cysteine residues through site-directed mutagenesis. Labeling of two mutants, gamma S8-C and gamma T106-C, in which Ser8 and Thr106, respectively, had been replaced by a cysteine, placed the gold cluster on the central mass that is seen in the hexagonal projection of the ECF1 complex. The results establish that the central mass contains the N-terminal part of the gamma subunit.
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PMID:Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy. 144 42

The structure of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using a variety of chemical and physiochemical techniques. The transmembrane topography of the H(+)-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H(+)-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H(+)-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H(+)-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H(+)-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH2- and COOH-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H(+)-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H(+)-ATPase based on this information and other considerations is presented.
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PMID:Probing the structure of the Neurospora crassa plasma membrane H(+)-ATPase. 146 Dec 58

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites. 153 26

The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.
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PMID:Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents. 153 73

The effects of two dibenzocyclooctene lignans on peroxidative damage of aging and ischemic rat brain were studied. Incubation of eight-month-old rat brain mitochondria and membrane suspension with Fe(2+)-cysteine resulted in the formation of malondialdehyde (MDA) and decrease of ATPase activity. Schisanhenol (Sal) (10(-4) M) completely inhibited the peroxidative damages of brain mitochondria and membrane of rats. The swelling and disintegration of brain mitochondria, as well as the reduction of brain membrane fluidity induced by Fe(2+)-cysteine were also prevented by Sal. The results of imitative experiment of ischemia and reperfusion of brain mitochondria and membrane in vitro indicated that Sal significantly impeded production of MDA and loss of ATPase activity induced by reoxygenation following anoxia. Oral administration of Sal induced increase of cytosol glutathione-peroxidase of brain in mice under the condition of reoxygenation following anoxia. The other compound schizandrin (Sin B) also has similar activity. But its potency is weaker than that of Sal. All these results indicate that Sal and Sin B have protective action against oxidative stress.
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PMID:Antioxidant activity of two dibenzocyclooctene lignans on the aged and ischemic brain in rats. 153 86

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzofurazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-Cl was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuriphenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethylmaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.
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PMID:Purification and properties of an ATPase from Sulfolobus solfataricus. 153 99

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide. 155 24


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