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)

In the present paper we report on an improved procedure for the preparation of free uterine cells which avoids the use of trypsin and employs very low concentration of collagenase. The cells released mechanically from the digested tissue are constantly removed from the enzyme containing medium, thus minimizing exposure to collagenase. 60%-70% of the cells which make up the intact uterus are obtained as free cells and 95% of these cells are viable for at least 15 hours at 37 degrees. Metabolic integrity was assessed by measuring the cell's ability to oxidize glucose and synthesize proteins over extended periods of time. The membrane leucine carrier protein and the membrane Na+/K+ ATPase were found to be fully functional. Electron microscopic analysis of the cells confirmed their structural integrity. Data are presented illustrating that with this system the estrogen binding protein is stable at physiological temperatures. The cells contain approximately 30,000 specific estrogen binding sites, with an apparent KA of 5--6 x 10(9) M-1. At 37 degrees 80% of the hormone receptor complexes were in the nuclear fraction, 20% in the cytoplasm. The similarity of the estrogen receptor binding parameters with those measured in the intact tissue after in vivo hormone adminsistration, together with the cells' structural and metabolic integrity make this procedure for the preparation of uterine cell suspensions in high yields particularly suitable for studies in which minimal cell injury is an essential prerequisite.
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PMID:An improved procedure for the preparation of rat uterine cell suspensions. 22 Jul 54

The estrogen receptor activation factor (E-RAF)-mediated binding of the receptor-estrogen complex to uterine nuclei was found to involve at least two classes of nuclear macromolecules: (1) the DNA, and (2) a proteinacious component. Evidences are presented to show that at least a portion of (2) is represented by the nuclear RNA polymerases. The receptor-estrogen complex associated in vivo with the nuclear RNA polymerases existed in two distinct forms which sedimented at 3.8 S and 4.8 S on sucrose density gradients. Almost 2/3 of the total radioactivity was associated with the 3.8 S species. Saturation kinetics of the two forms showed that while the 4.8 S form displayed characteristics similar to the classical type I nuclear binding site, the features displayed by the 3.8 S form were closely similar to those of the nuclear type II site. The 4.8 S species is a DNA binding form while the 3.8 S form is non-DNA binding. Anti-E-RAF IIA IgG cross-reacted with both the binding components. Goat uterine E-RAF I, IIA and IIB were purified to homogeneity as described earlier. While E-RAF IIA and IIB destabilized the native DNA structure and induced separation of the DNA strands, E-RAF I performed the opposite function. The reactions required the presence of ATP; all three of them displayed DNA-dependent ATPase activity. In an in vitro transcription system which contained purified RNA polymerase B (rat liver enzyme) and goat uterine DNA, E-RAF IIA and IIB enhanced transcription 7-fold over the control while E-RAF I totally suppressed the transcription process, irrespective of whether it was stimulated earlier by the other two E-RAF forms or not. This E-RAF property remained unchanged even after its association with the 4 S receptor-estrogen complex forming 5 S complex.
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PMID:Molecular aspects of estrogen receptor activation factor (E-RAF) function. 252 74

The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173-250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323-395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action.
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PMID:Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors. 302 Nov 26

A 55-kDa protein (p55) that mediates the transport of the estrogen receptor (ER) from the cytoplasm to the nucleus has been purified to homogeneity from goat uterine cytosol. Using this pure protein, some of the aspects of the mechanisms associated with the transport of the ER to the nucleus have been elucidated. The mechanism of ER transport into the nucleus can be separated into two steps: the p55-mediated transport and binding of ER to the nuclear membrane, followed by an ATP-dependent, 14-kDa protein(s)-mediated translocation of ER into the nucleus. The p55 has inherent ATPase activity and it is proposed that the energy released during this ATP hydrolysis is utilized in the nuclear transport of the ER.
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PMID:A 55-kDa protein (p55) of the goat uterus mediates nuclear transport of the estrogen receptor. II. Details of the transport mechanism. 778 42

Many constitutional analogues of estrogen have been reported. In this review, some new synthetic estrogens possessing a different action from natural estrogens are described. Aminoestrogens inhibit platelet aggregation. Estradiol-chlorambucil conjugate (KM2210) decreases the level of estrogen receptor mRNA in the human breast cancer cell MCF-7. The representative synthetic estrogen, diethylstilbestrol, and its derivatives show an inhibitory action against H(+)- and Ca(2+)-ATPase. Moreover, the effect of the diastereometric [1,2-bis(2,6-dihalo-3-hydroxyphenyl)ethylenediamine ] sulfatoplatinum (II) complexes, derivatives of hexesterol-platinum complexes, on estrogenic action and the growth-inhibitory action of hormone dependent breast cancer, are strongly dependent on the halogen atoms and on their position in the aromatic ring. These drugs are expected to be applicable for clinical use.
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PMID:[Synthetic estrogens--new pharmacological actions and mechanisms]. 816 54

The human SNF2alpha (or hbrm) and SNF2beta (or BRG1) proteins have previously been shown to enhance transcriptional activation by nuclear receptors (NRs) in cultured human cells, and to be present in SWI/SNF complexes which are thought to be involved in control of transcription by facilitating remodelling of chromatin templates. Using the yeast two-hybrid system, we now demonstrate that the N-terminal regions of hSNF2alpha and hSNF2beta, preceding the DNA-dependent ATPase domain, specifically interact with the region of the estrogen receptor (ER) which includes the ligand binding domain and the ligand-dependent activation function AF-2. These interactions are increased by estrogen, but not by the ER AF-2 antagonist hydroxytamoxifen. Furthermore, mutants of ER that lack AF-2 activity are unable to interact with hSNF2alpha and -beta. These results suggest that the human homologues of the yeast SWI2/SNF2 protein may participate in the enhancement of transcription by the ER in vivo through interactions with the AF-2 activating domain, thus leading to ligand-dependent remodelling of chromatin templates.
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PMID:Ligand-dependent interaction between the estrogen receptor and the human homologues of SWI2/SNF2. 909 65

The extracellular pH in malignant tumors is known to be lower than in normal tissues and may therefore facilitate extracellular activation of secreted lysosomal cathepsins. We have tested the capability of human mammary cells (continuous cell lines and primary culture) to acidify their extracellular environment, using two techniques. By measuring pH changes through alterations of phenolsulfone phthaleine absorbance, we found that the more aggressive MDA-MB-231 human breast cancer cells were more active in acidifying a non-buffered balanced salt solution than the estrogen receptor positive MCF7 and ZR75 cell lines and than normal mammary epithelial cells in primary culture. Metastatic breast cancer cells from pleural effusions were up to 200-fold more active in acidifying their extracellular milieu than non-malignant mammary cells cultured in the same conditions, strongly suggesting that this difference also occurs in vivo. The use of inhibitors in the presence or absence of glucose showed that both lactate and an ATP-driven proton pump sharing some characteristics of the vacuolar H+ pump were involved. Bafilomycin A1, a specific inhibitor of the vacuolar (V-type) ATP-H+ pump inhibited part of the acidification by MCF7 cells, but not by MDA-MB-231 cells. We also used microelectrodes to measure extracellular pH, in close contact to the MCF7 breast cancer cells. The pH at the free surface of MCF7 cells was lower by 0.33 +/- 0.14 unit than that of the surrounding medium, while insertion of the microelectrode tip beneath the attached surface of the cells showed a greater lowering of pH from 0.3 to 1.7 pH unit as long as cell attachment on the substrate prevented H+ diffusion. We conclude that breast carcinoma cells have a higher capacity for acidifying their extracellular milieu than normal mammary cells, and that both a plasma membrane H(+)-ATPase, and lactic acid production are involved in this acidification. It is therefore possible that the aspartyl and cysteinyl pro-cathepsins secreted in excess by tumor cells may be activated extracellularly in vivo close to the basement membrane.
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PMID:Breast cancer cells have a high capacity to acidify extracellular milieu by a dual mechanism. 921 26

Tamoxifen has been reported to inhibit acidification of cytoplasmic organelles in mammalian cells. Here, the mechanism of this inhibition is investigated using in vitro assays on isolated organelles and liposomes. Tamoxifen inhibited ATP-dependent acidification in organelles from a variety of sources, including isolated microsomes from mammalian cells, vacuoles from Saccharomyces cerevisiae, and inverted membrane vesicles from Escherichia coli. Tamoxifen increased the ATPase activity of the vacuolar proton ATPase but decreased the membrane potential (Vm) generated by this proton pump, suggesting that tamoxifen may act by increasing proton permeability. In liposomes, tamoxifen increased the rate of pH dissipation. Studies comparing the effect of tamoxifen on pH gradients using different salt conditions and with other known ionophores suggest that tamoxifen affects transmembrane pH through two independent mechanisms. First, as a lipophilic weak base, it partitions into acidic vesicles, resulting in rapid neutralization. Second, it mediates coupled, electroneutral transport of proton or hydroxide with chloride. An understanding of the biochemical mechanism(s) for the effects of tamoxifen that are independent of the estrogen receptor could contribute to predicting side effects of tamoxifen and in designing screens to select for estrogen-receptor antagonists without these side effects.
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PMID:A mechanism for tamoxifen-mediated inhibition of acidification. 1037 41

An expedient method for the purification of a calcium activated neutral protease (CANP) of the goat uterus has been designed. This enzyme, purified to homogeneity, has been used as a tool in the structural characterization of the estrogen receptor activation factor II (E-RAF II) that dimerizes with an alternative form of estrogen receptor (ER), the non-activated estrogen receptor (naER). The enzyme cleaves the E-RAF into two fragments, alpha and beta, of molecular mass 32 and 30 kDa, respectively. The beta retains the DNA binding activity, as well as the capacity to dimerize with the naER. On the other hand, the cholesterol binding activity and the ATPase function are shared by both alpha and beta fragments. The E-RAF domain that binds to the nuclear periphery appears to be localized on the beta fragment. The beta fragment, however, is incapable of entering the nucleus on its own.
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PMID:Structural characterization of the goat uterine estrogen receptor activation factor using an endogenous calcium activated neutral protease. 1043 23

A 55 kDa nuclear localization signal binding protein (p55) is involved in the transport of the goat uterine estrogen receptor from the cytoplasm to the nuclear pore complex (NPC). p55 forms a complex with a 12 kDa protein (p12) which in turn becomes 'docked' at the NPC. The present study reports on the purification and functional characterization of p12. Both p55 and p12 are Mg2+-dependent ATPases. The protein-protein interactions that take place between these two molecules at the NPC cause an enhancement in the net ATPase activity associated with the protein complex. Presumably, this enhanced ATPase function helps in the final nuclear entry of the estrogen receptor; p55 remains associated with p12 at the nuclear entry site under these conditions.
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PMID:Interdependence between a 55 kDa protein (p55) and a 12 kDa protein (p12) in facilitating the nuclear entry of goat uterine estrogen receptor under cell-free conditions. 1083 56


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