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)

We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.
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PMID:Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase. 131 20

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.
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PMID:Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences. 133 74

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.
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PMID:Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles. 153 18

Transplasm membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes contain b cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na+/H+ antiport. In plants part of the proton release can be achieved by activation of the H+ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.
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PMID:Electron and proton transport across the plasma membrane. 172 Oct 49

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.
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PMID:Two types of clathrin-coated vesicles isolated from rat brain: analysis of biochemical properties and cellular origin. 184 49

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.
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PMID:Isolation of functional, coated, endocytic vesicles. 190 Mar 1

Some indices of nonspecific reactivity: the number of cyclic immunological complexes, titer of antibodies, activity of alkaline and acid phosphatases, alanine aminotranspherase, mitochondrial liver H(+)-ATPase, content of glutathione ceruloplasmin transferrin are stated to change in the model of food anaphylaxis in 37 guinea pigs, 26 rats and 12 rabbits orally sensitized with hen egg protein and they are estimated prior to and after the allergy development.
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PMID:[Nonspecific allergy indices in oral sensitization by chicken egg white]. 205 32

Internalization of EGF and transferrin measured as the rate of uptake of 125I-labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS-120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS-120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I-EGF and 125I-transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL-39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I-EGF to CCL-39 and PS-120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL-39 or PS-120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+-ATPase. Ouabain, an inhibitor of Na+, K+-ATPases, showed no effect on 125I-EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane-bound Na+/H+ antiport, a major pHi-regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.
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PMID:The role of intracellular pH in ligand internalization. 242 21

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.
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PMID:Uptake and subcellular distribution of injected transferrin in rat liver. 244 12

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.
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PMID:Regulation of endocytic pH by the Na+,K+-ATPase in living cells. 253 68


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