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)

The characteristics of the interaction between N-(3-pyrene)maleimide (PMI) and the sarcoplasmic reticulum (SR) membranes were investigated, and the sites of labeling with PMI on Ca(2+)-transporting ATPase were identified. PMI was dissolved in the membrane lipids before reacting with the ATPase protein. The measurement of resonance energy transfer from PMI to 1-(dimethylaminophenyl)-6-phenyl-1,3,5-hexatriene revealed that the pyrene moiety of PMI stayed in the lipid layer after it had been covalently attached to the ATPase molecule. PMI-labeled SR membranes at an average labeling density of 1 mol PMI/mol ATPase were digested with trypsin, and the labeled peptides were purified through a series of reversed-phase HPLC procedures on C18 and C4 columns. The amino acid analysis of the purified peptides revealed multiple cysteine residues mainly distributed over the C-terminal half of the cytoplasmic domain of the ATPase molecule as the targets of PMI. This implied that PMI molecules mediated cross-linking between the cytoplasmic domain of the ATPase molecule and the membranes. The distortion of the structure of the former due to this cross-linking may explain the uncoupling of ATP-splitting from Ca(2+)-transport caused by PMI.
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PMID:Sites of labeling with N-(3-pyrene)maleimide on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum. 759 54

Polycyclic aromatic hydrocarbons (PAHs) are immunosuppressive chemicals found in the environment that have been shown to disrupt intracellular Ca2+ homeostasis and Ca(2+)-dependent signaling in human and murine lymphocytes. Many PAHs produce a rapid and sustained increase in intracellular free Ca2+ in lymphocytes. The mechanism of persistent Ca2+ perturbation remains undefined. In the present studies, ATP-dependent 44Ca2+ uptake into vesicles prepared from a 15,000g supernatant of HPB-ALL human T cell lysates was significantly inhibited by 0.1, 1, and 10 microM concentrations of the immunotoxic PAHs 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BAP), benz[a]anthracene, and 9,10-dimethylanthracene, but not by the less immunotoxic compounds anthracene (ANT) and benzo[e]pyrene (BEP). Ca(2+)-ATPase catalytic activity was determined by quantitating hydrolysis of ATP in the presence or absence of PAHs, with known ATPase inhibitors included as controls. Formation of inorganic phosphate was significantly decreased (> 65% of control at 10 microM) by DMBA and BAP, whereas ANT and BEP caused only a slight reduction in activity (10% of control at 10 microM). Anthracene partially reversed the inhibitory effect of DMBA and BAP on ATP hydrolysis when agents were coincubated. Both DMBA and BAP, but not ANT and BEP, inhibited the activity of all known SERCA-type Ca(2+)-ATPases, while not affecting either Na+, K(+)-ATPase activity or plasma membrane Ca(2+)-ATPase activities. These results demonstrate that immunotoxic and carcinogenic polycyclic aromatic hydrocarbons have a thapsigargin-like effect in human lymphocytes and SERCA-containing tissues from various species. Inhibition of SERCA activity may play an important role in altered Ca2+ homeostasis in lymphocytes and other tissues.
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PMID:Inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons in HPB-ALL human T cells and other tissues. 759 99

The cells derived from the human embryo liver tissue were transfected with a plasmid pSV3neo containing both the large and small T-antigen gene of the early region of simian virus 40 (SV40), and two cell strains, OUMS-21 and -22, were obtained. OUMS-22 cells, to date, have reached over 100 population doublings through a culture crisis and are considered to have become an immortal cell line. However, OUMS-21 cells failed to become an immortal cell line. Both OUMS-21 and -22 cells were SV40 T-antigen-positive, epithelial-like, and immunoreactive against an anti-keratin 18 monoclonal antibody but against neither an anti-vimentin nor an anti-von Willebrandt factor VIII monoclonal antibody. The staining pattern of cytokeratin in these cells was similar to that in the differentiated human hepatoblastoma and hepatocellular carcinoma cell lines but not to that in the human cholangiocellular carcinoma cell lines. OUMS-21 and -22 cells expressed neither alpha-fetoprotein nor albumin mRNAs. These cells showed no tyrosine aminotransferase activity. However, both OUMS-21 and -22 cells were sensitive to cytotoxicity of aflatoxin B1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, and benzo[a]pyrene, whereas human embryo lung fibroblasts were insensitive to the cytotoxicity of these carcinogens. These findings suggest that OUMS-21 and -22 cells may arise from undifferentiated liver stem cells or from hepatocytes that lost their ability to express the liver-specific functions prior to immortalization. Both OUMS-21 and -22 cells expressed glutathione S-transferase pi (GST-pi) mRNA. The expression of GST-pi mRNA highly increased in OUMS-22 cells with their immortalization. Karyotypic analysis showed that numerical and structural aberrations of the chromosomes were profound, but neither specific events nor marker chromosomes were found in OUMS-21 and -22 cells. Both OUMS-21 and -22 cells could grow in soft agar, but they were not tumorigenic when transplanted into nude mice.
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PMID:Immortalization of epithelial-like cells from human liver tissue with SV40 T-antigen gene. 768 77

Testosterone, testosterone propionate, 17 beta-trenbolone and progesterone, which represent the main endogenous and synthetic androgens and a progestin, were evaluated for possible cell transformation and genetic effects in Syrian hamster embryo (SHE) cells. Cell growth was reduced by treatment with the steroids at 10-30 micrograms/ml in a dose-related manner. Testosterone and testosterone propionate were less toxic than the other two steroids. Testosterone, testosterone propionate and progesterone induced morphological transformation of SHE cells with similar transformation frequencies. The most potent effects were observed with testosterone propionate, which induced cell transformation at 1-30 micrograms/ml in a dose-related manner. Testosterone and progesterone transformed cells only at the highest dose (30 micrograms/ml). 17 beta-Trenbolone did not induce a statistically significant level of cell transformations at any dose tested (up to 30 micrograms/ml). The transformation frequencies induced by testosterone, testosterone propionate and progesterone were less than one-half that induced by benzo[a]pyrene at 1 microgram/ml. None of these steroids induced significant increases in frequencies of chromosome aberrations or aneuploidy. Gene mutations were not observed for testosterone at the HPRT or Na+/K+ ATPase locus. Because these steroids are also associated with carcinogenic activity in vivo, these in vitro findings provide a model and new insights into the study of the mechanisms of androgen- and progestin-induced cell transformation.
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PMID:Effects of testosterone, testosterone propionate, 17 beta-trenbolone and progesterone on cell transformation and mutagenesis in Syrian hamster embryo cells. 778 50

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.
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PMID:Structural connectivity in actin: effect of C-terminal modifications on the properties of actin. 785 32

Experimental evidence for the involvement of the 18-29 site within actin subdomain-1 in the actomyosin weak binding interface includes the inhibition of actomyosin ATPase activity by specific peptide antibodies [Adams, S., & Reisler, E. (1993) Biochemistry 32, 5051-5056] and by the Dictyostelium actin mutant D24H/D25H [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In this work, the effect of the 18-29 peptide antibodies on the polymerization and conformation of actin has been characterized. Binding of antibody to the 18-29 site strongly inhibited the MgCl2-induced polymerization of G-actin, had a much weaker impact on the CaCl2 polymerization of actin, and showed very little effect on the NaCl polymerization of G-actin. These observations were linked to the binding of the 18-29 antibody to the different forms of actin. In sedimentation assays, the (18-29) IgG bound more strongly to Mg-F- and Mg-G-actins than to Ca-F- and Ca-G-actins, respectively. The binding of IgG to F-actin decreased sharply with an increase in ionic strength. Antibody binding to the 18-29 site induced conformational changes within the nucleotide cleft, both slowing the rate of nucleotide exchange and increasing the fluorescence intensity of actin-bound epsilon ATP. The increased fluorescence of a dansyl probe attached to Gln-41 and a pyrene probe attached to Cys-374 demonstrated that antibody binding also caused local perturbations in the DNase I loop of subdomain-2 and at the C-terminus of actin. These results are discussed in terms of actin plasticity and its implications for actomyosin interactions.
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PMID:Sequence 18-29 on actin: antibody and spectroscopic probing of conformational changes. 798 Dec 2

The kinetic mechanism of myofibril ATPase was investigated using psoas and mixed back muscle over a range of ionic strengths. Myofibrils were labeled with pyrene iodoacetamide to measure the rate constants for the binding of ATP and formation of the weakly attached state. The velocity of shortening was measured by stopping the contraction at various times by mixing with pH 4.5 buffer. The transient and steady-state rates of ATP hydrolysis were measured by the quench flow method. The results fitted the kinetic scheme [formula: see text] The rate constants (or equilibrium constants for steps 1 and 6) were obtained for the six steps. k5 was calculated from the KM for shortening velocity, K1, and k2. The rate constants were essentially equal for myofibrils and acto-S-1 at low ionic strength. Increasing the ionic strength up to 100 mM in NaCl increased the rate of the hydrolysis step and the size of the phosphate burst and the effective rate of product release became the rate-limiting step. The step calculated from the velocity of shortening, k5, and k2 is 15 nm, based on a model in which step 4 is the force-generating step.
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PMID:Kinetic mechanism of myofibril ATPase. 806 Dec 3

The sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle were allowed to react with N-(3-pyrene)maleimide (PMI) at pH 7 at 30 degrees C. The Ca(2+)-transporting activity of the SR membranes was reduced to 20% when PMI was bound to the extent of 1 mol/mol of Ca(2+)-transporting ATPase. The ATPase and the E-P forming activities were not affected by the binding of PMI up to 2 mol/mol ATPase, indicating that PMI somehow uncoupled Ca(2+)-transport from ATP splitting. Permeability of the SR membranes to Ca2+ ions was increased in parallel with the loss of the Ca(2+)-transporting activity. Of several components of the SR membranes which are reactive with PMI, the ATPase protein was the only one whose modification by PMI was directly related to the loss of the Ca(2+)-transporting activity. Similar results were obtained with the light SR membrane fraction, which lacks the ryanodine receptor, a well-recognized Ca2+ channel. These results indicated that a Ca2+ channel that would have been latent or properly regulated in native ATPase somehow escaped from the normal control mechanism as a result of modification of its SH groups by PMI and went into runaway operation. The activated channel was specific for alkaline earth metal ions, so permeability to other solutes including Co2+, Ni2+, and sucrose remained unchanged after treatment with PMI.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Uncoupling of ATP splitting from Ca(2+)-transport in Ca(2+)-transporting ATPase of the sarcoplasmic reticulum as a result of modification by N-(3-pyrene)maleimide: activation of a channel with a specificity for alkaline earth metal ions. 826

The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 by maleimides such as N-ethylmaleimide or fluorescein maleimide resulted in a reconstitutively active enzyme that was indistinguishable from the native protein. Labeling of Cys64 with or without concomitant labeling of Cys140 resulted in a reconstitutively inactive enzyme. The ATPase activity of either form of the labeled enzyme was unaffected. However, labeling of Cys64 was accompanied by dissociation of the delta subunit from the enzyme. These observations suggest a role for the microenvironment of Cys64 in interactions of the delta subunit with other subunits in the enzyme. Two types of evidence support the conclusion that the 2 cysteine residues of the delta subunit are in close proximity. First, incorporation of pyrene maleimide into both delta cysteines led to excimer formation. Second, incubation of F1 with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in quantitative formation of a disulfide bond between Cys64 and Cys140, presumably via disulfide interchange. The enzyme containing the internally cross-linked delta subunit exhibited an undiminished ability to support proton pumping when reconstituted into F1-depleted membrane vesicles. The presence of 2 closely apposed cysteinyl residues in the delta subunit of the native enzyme places constraints on the type of structure that may be proposed for the subunit.
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PMID:Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase. 830 87

During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. To investigate the protein-protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N-terminal 42 kDa ATPase region and the C-terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34. 840 47


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