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)

Synthetic mRNAs (i.e. cRNA alpha and cRNA beta) were obtained by cell-free transcription of M13 KS(+) (Bluescript) expression vectors which contained the entire coding region of the alpha or beta subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA alpha yielded full length alpha polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNA beta yielded a single immunoprecipitable full length polypeptide. Association of the alpha polypeptide with the microsomal membranes was obtained only co-translationally. Fifteen to 50% of the membrane-associated alpha subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the alpha subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the beta polypeptide was glycosylated as indicated by the appearance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The beta subunit was predominantly translocated into the lumen of the endoplasmic reticulum since 90% of the mass of the membrane-associated beta polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.5 kDa), and 95% of all of the beta chains were resistant to extraction with alkali. Neither the alpha nor the beta subunits have NH2-terminal leader signal sequences, but both may require the signal recognition receptor for membrane insertion, as evidenced by inhibition of incorporation of both subunits into microsomes pretreated with N-ethylmaleimide. Simultaneous translation of cRNA alpha and cRNA beta did not enhance membrane insertion of either the alpha or beta polypeptide.
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PMID:Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit cDNAs. 169 72

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase. 254 51

The plasma membrane H+-ATPase of Neurospora is a 100-kDa integral membrane protein which appears, on the basis of hydropathy analysis of its amino acid sequence, to span the lipid bilayer at least eight times. To investigate the assembly and processing of the ATPase, a full-length cDNA has been constructed for use in in vitro transcription and translation experiments. Comparison of three different forms of the ATPase (nascent protein, nascent protein cotranslationally inserted into membranes, and mature protein) revealed no difference in electrophoretic mobility. Furthermore, the nascent and mature forms gave identical peptide patterns after partial proteolysis with Staphylococcus aureus V8 protease, suggesting that the ATPase does not contain an NH2-terminal signal peptide which is cleaved upon membrane insertion. Consistent with this interpretation, the NH2-terminal peptide has been purified from a tryptic digest of the ATPase and found to lack only the initiator methionine residue; the penultimate alanine is acetylated based on analysis by fast atom bombardment mass spectroscopy. Although the ATPase contains one potential site of N-linked glycosylation, its electrophoretic mobility was unchanged following digestion with endoglycosidase H and it did not incorporate [3H]mannose or bind concanavalin A. Thus, the Neurospora plasma membrane-ATPase appears to undergo minimal post-translational processing, and its membrane insertion is probably mediated by internal sequences.
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PMID:Biosynthesis of the plasma membrane H+-ATPase of Neurospora crassa. 290 84

The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse-chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h).
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PMID:The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport. 300 48

A 174-kilodalton polypeptide of the Drosophila nuclear matrix-pore complex-lamina fraction has been identified as an ATPase/dATPase on the basis of direct UV photoaffinity labeling studies; a polypeptide with similar properties has been found in nuclear envelope fractions prepared from several vertebrate sources (Berrios, M., Blobel, G., and Fisher, P. A. (1983) J. Biol. Chem. 258, 4548-4555). This ATPase/dATPase polypeptide co-migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels with a glycoprotein also found in all of these fractions. In rat liver, this glycoprotein has been localized to the nuclear pore complex by means of immunoelectron microscopy (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837). Following SDS denaturation and reduction/alkylation, chromatography of the Drosophila nuclear matrix-pore complex-lamina fraction on hydroxylapatite columns run in the presence of SDS results in the separation of two quantitatively major 174-kilodalton polypeptides. The peak of glycoprotein elution from the SDS-hydroxylapatite column correlates exactly with that of the early eluting 174-kilodalton species while the photolabeled ATPase/dATPase polypeptide co-chromatographs with the late eluting one. Identical results have been obtained with the rat liver nuclear envelope fraction. The chromatographically separated 174-kilodalton species from both organisms have been further distinguished through the use of polypeptide-specific antisera; finally, the glycoprotein purified from Drosophila embryos is fully sensitive to limited degradation by endoglycosidase H whereas the ATPase/dATPase polypeptide is completely resistant. We have thus established, using material obtained from two widely divergent higher eukaryotes, that the 174-kilodalton ATPase/dATPase is a quantitatively major nuclear matrix-pore complex-lamina component distinct from the nuclear pore complex glycoprotein of apparently identical molecular weight.
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PMID:A 174-kilodalton ATPase/dATPase polypeptide and a glycoprotein of apparently identical molecular weight are common but distinct components of higher eukaryotic nuclear structural protein subfractions. 613 58

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.
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PMID:The yeast proprotein convertase encoded by YAP3 is a glycophosphatidylinositol-anchored protein that localizes to the plasma membrane. 765 70

The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by 36Cl- uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same "double-barreled" gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived sequence. This "ClC-0" protein is lightly glycosylated, losing only approximately 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-0 channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (approximately equal to 150 kDa) and substantially smaller than the reduced form of the nicotinic acetylcholine receptor (approximately equal to 300 kDa). The detergent-solubilized ClC-0 channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.
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PMID:Purification, reconstitution, and subunit composition of a voltage-gated chloride channel from Torpedo electroplax. 794 26

The secretory form of IgM (sIgM) undergoes developmentally regulated intracellular sorting and transport, as demonstrated by the distinct assembly patterns and intracellular fate exhibited by its mu heavy chain (microseconds). In the 38C B lymphocytes, microseconds-containing monomers are retained and degraded intracellularly, whereas in the 38C-derived D2 hybridoma, microseconds-containing polymers are secreted. Here we show that sorting of sIgM is impaired in the presence of the thiol-reducing agent beta-mercaptoethanol or when cellular calcium sequestration is perturbed either with the Ca2+ ionophore A23187 or with thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Under these conditions, sIgM evades retention/degradation in the 38C cells, resulting in its enhanced secretion. Conversely, in the D2 cells, secretion is strongly attenuated, and sIgM is partly degraded. Both cell types secrete unusually processed sIgM, which is completely resistant to endoglycosidase H, unlike the partially sensitive sIgM secreted in the usual manner, indicating terminal glycosylation of all carbohydrate moieties of microseconds. Moreover, secretion is no longer restricted to fully assembled polymeric sIgM, and in both cell types under Ca2+ perturbation preferentially hemimeric and monomeric assembly intermediates are detected in the medium. Our results suggest that the intracellular fate of sIgM is collectively determined by several sorting events that confer sIgM retention/degradation, ensure sIgM secretion from D2 cells, and prevent the arrival of assembly intermediates to the cell surface. All of these sorting events appear to require high Ca2+ concentration and oxidizing thiol redox state, the conditions that prevail in the lumen of the endoplasmic reticulum. Thus, thiol and calcium are implicated as modulators of intracellular traffic.
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PMID:Thiol-reducing agents and calcium perturbants alter intracellular sorting of immunoglobulin M. 796 45

Folimycin (concanamycin A) specifically inhibited vacuolar-type ATPase as far as examined. Folimycin blocked excretion of the glycoprotein (G protein) of vesicular stomatitis virus into the medium and, instead, G protein was accumulated intracellularly. The intracellularly accumulated G protein electrophoresed a little faster than mature one. The N-glycan of the G protein was endoglycosidase H-sensitive, and terminal galactose and N-acetylglucosamine were not detected essentially on sequential digestion with exoglycosidases, indicating that processings known to occur in the Golgi apparatus do not take place in the presence of folimycin. The oligosaccharide chain of the G protein was determined to have a composition of Man8GlcNAc2 as analyzed by Bio-Gel P-4 column chromatography and high-performance liquid chromatography following digestion with alpha- and then with beta-mannosidase. Activities of mannosidase I and glycosyltransferases prepared from baby hamster kidney cells were not inhibited as far as examined, indicating that the incompleteness of the N-glycosidic chain in folimycin-treated cells is not caused by inhibition of processing enzymes. Taken together these observations suggest that folimycin blocks the intracellular translocation of G protein before the step of trimming by mannosidase I which is confined to the cis compartment of the Golgi. The intracellular localization of G protein as revealed by fluorescence microscopy was in good accordance with this assumption.
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PMID:Folimycin (concanamycin A), a specific inhibitor of V-ATPase, blocks intracellular translocation of the glycoprotein of vesicular stomatitis virus before arrival to the Golgi apparatus. 824 93

The gastric proton pump, H(+)-K(+)-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K(+)-stimulated ATP hydrolysis as well as K(+)-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H(+)-K(+)-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.
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PMID:Characterization of the beta-subunit of the H(+)-K(+)-ATPase using an inhibitory monoclonal antibody. 827 17


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