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

By altering the Na+/K+ electrochemical gradient, Na+,K(+)-ATPase activity profoundly influences cardiac cell excitability and contractility. The recent finding of mineralocorticoid hormone receptors in the heart implies that Na+,K(+)-ATPase gene expression, and hence cardiac function, is regulated by aldosterone, a corticosteroid hormone associated with certain forms of hypertension and classically involved in regulating Na+,K(+)-ATPase gene expression and transepithelial Na+ transport in tissues such as the kidney. The regulation by aldosterone of the major cardiac Na+,K(+)-ATPase isoform genes, alpha-1 and beta-1, were studied in adult and neonatal rat ventricular cardiocytes grown in defined serum-free media. In both cell types, aldosterone-induced a rapid and sustained 3-fold induction in alpha-1 mRNA accumulation within 6 h. beta-1 mRNA was similarly induced. alpha-1 mRNA induction occurred over the physiological range with an EC50 of 1-2 nM, consistent with binding of aldosterone to the high affinity mineralocorticoid hormone receptor. In adult cardiocytes, this was associated with a 36% increase in alpha subunit protein accumulation and an increase in Na(+)-K(+)-ATPase transport activity. Aldosterone did not alter the 3-h half-life of alpha-1 mRNA, indicating an induction of alpha-1 mRNA synthesis. Aldosterone-dependent alpha-1 mRNA accumulation was not blocked by the protein synthesis inhibitor cycloheximide, whereas amiloride inhibited both an aldosterone-dependent increase in intracellular Na+ [Na+]i) and alpha-1 mRNA accumulation. This demonstrates that aldosterone directly stimulates Na+,K(+)-ATPase alpha-1 subunit mRNA synthesis and protein accumulation in cardiac cells throughout development and suggests that the heart is a mineralocorticoid-responsive organ. An early increase in [Na+]i may be a proximal event in the mediation of the hormone effect.
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PMID:Aldosterone-mediated regulation of Na+, K(+)-ATPase gene expression in adult and neonatal rat cardiocytes. 164 19

Mastoparan is a 14-amino-acid peptide that stimulates secretion from several cell types. Secretion can be partially blocked by pertussis toxin and may be mediated by guanine-nucleotide-binding proteins (G-proteins). Mastoparan can act directly on G-proteins, probably at the hormone receptor-binding site, to stimulate guanosine 5'-[gamma-thio]triphosphate binding and GTPase activities of pertussis-toxin substrates Go and Gi [Higashijima, Uzu, Nakajima & Ross (1988) J. Biol. Chem. 263, 6491-6494]. We now describe a nucleotidase from bovine brain that is not a known G-protein whose GTPase and ATPase activities are stimulated by mastoparan. This nucleotidase hydrolyses ATP faster than GTP, but has similar affinities for both (0.4 microM). Mastoparan maximally stimulates both ATPase and GTPase activities by about 8-fold after insertion of the protein into phospholipid vesicles, but does not affect the EC50 (concentration at which half the maximal effect is observed) for ATP and GTP. The EC50 for mastoparan stimulation of GTPase and ATPase is 6 and 12 microM respectively. The native molecular mass of the partially purified mastoparan-stimulated nucleotidase is 87 kDa. This nucleotidase may be another receptor-activated enzyme, and its identification may be useful for understanding mastoparan-stimulated processes.
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PMID:Characterization of a mastoparan-stimulated nucleotidase from bovine brain. 165 78

We have determined the sequence of the 5'-flanking region and first three exons of the human Na,K-ATPase alpha 1 gene, ATP1A1. Primer extension and S1 nuclease protection analyses of RNA from human kidney, brain, and skeletal muscle indicate that transcription initiates 273 nucleotides upstream of the translation start site. The promoter region contains a potential TATA box at position -27 relative to the transcription initiation site; however, no CCAAT sequence is observed. The 5'-untranslated and 5'-flanking regions are G + C rich. Five sequence elements exhibiting similarity to binding sites for the transcription factor Sp1 are located within the 5'-flanking region. This region also contains potential binding sites for the transcription factors AP-1, AP-2, AP-3, and NF-1, as well as a site which exhibits perfect identity to an 8-bp sequence element important for calcium induction. A comparison of the 5'-flanking region of the alpha 1 and alpha 2 genes reveals differences in potential transcription factor and hormone receptor binding sites which may be important in mediating the tissue- and developmental stage-specific expression of these genes. We have also identified an intragenic DNA probe which detects a restriction fragment length polymorphism at the alpha 1 locus. This marker should facilitate genetic linkage studies designed to evaluate the role of the sodium pump in human disease.
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PMID:The human Na, K-ATPase alpha 1 gene: characterization of the 5'-flanking region and identification of a restriction fragment length polymorphism. 197 Mar 26

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.
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PMID:Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles. 217 38

Our prior studies with inside-out plasma membrane vesicles from L1210 cells (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) identified an outwardly directed, translocating ATPase as mediating the majority of methotrexate (MTX) efflux in these cells. In the current studies, we examined by competitive inhibition with [3H]MTX as permeant some of the structural features that determine preferences among folate compounds and their analogues as permeants for this ATPase. The results show that folate compounds are preferred over simple quinazolines (5,8-dideaza-pteridines), and IL5-CH3-folateH4, and probably other 5-substituted folates are preferred over folic acid. In the latter regard, the observed equivalence in preference to IL5-CH3-folateH4 of the 4-oxa-pyridopyrimidine, lometrexol (DDATHF), probably relates to its close similarity to folateH4. The results also suggest that the 4-position in the case of folate analogues, but not in the case of the quinazoline analogues, is an important determinant with 4-amino preferred over 4-oxa. They also suggest that the N10 position on the bridge region in both series of compounds, and probably for the pyridopyrimidine lometrexol, is not an important determinant. In contrast to results seen with the simple quinazolines, the 2-CH3 desamino quinazoline ZEN D1694, modified as well by a 2-benzyl to thienyl replacement on the side chain, was highly preferred. The same relative differences seen among some of these analogues as inhibitors of [3H]MTX efflux in inside-out vesicles were documented for their effectiveness as permeants for ATP-dependent efflux in intact L1210 cells.
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PMID:Structural preferences among folate compounds and their analogues for ATPase-mediated efflux by inside-out plasma membrane vesicles derived from L1210 cells. 776 86

The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation.
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PMID:SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals. 863 5

Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR). Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP). However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1. The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line. Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells. This result suggests that these modulators exert their effect by directly interacting with P-gp. Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5. This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency. Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring. Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells. The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3. These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.
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PMID:Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells. 961 55

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.
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PMID:Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects. 975 79

Sodium-water balance is causally linked to the functional expression of a number of important ocular tissues, viz. corneal deturgescence, aqueous humor secretion by the iris, hydration of the lens, retinal photoreception, and choriocapillary angiogenesis. The regulation of sodium absorption in the eye is generally believed to be under the control of Na(+),K(+)-activated adenosine triphosphatase, although evidence for this view is at best circumstantial. Contemporary work has shown widespread distribution of the mineralocorticoid hormone receptor and its colocalization with the amiloride-sensitive sodium channel in cells of diverse embryological origins. All available evidence favors the idea that the transcriptional regulation of the apical sodium channel by adrenocorticoids, and not the basolateral sodium pump, is critically important to sodium-water homeostasis in various ocular tissues, in a manner previously believed to be limited exclusively to the epithelial cell in various peripheral organs. Based upon these parameters, models are presented to help in understanding the direction of sodium absorption in a number of ocular tissues. Thus, the regulation of the sodium channel by steroid hormones seems to be a universal feature of the living cell that may have important implications in the understanding and management of normal ocular functions and their modification in human pathology.
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PMID:Receptor-mediated adrenocorticoid hormone signaling in ocular tissues. 1269 62


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