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)

Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of 45Ca2+ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of 45Ca2+ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca(2+)-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca(2+)-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.
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PMID:Several compartments of Saccharomyces cerevisiae are equipped with Ca(2+)-ATPase(s) 820 May 6

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.
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PMID:Biochemical characterization and intracellular localization of the Menkes disease protein. 894 55

The Na+/K+ ATPase is composed of two subunits called alpha and beta chains. In insect cells, independently expressed alpha and beta chains are localized to intracellular membranes. Sucrose density gradient sedimentation, crosslinking analysis, and immunoprecipitation of radio-labeled proteins show that the alpha chains expressed alone are in large aggregates of different molecular weights with less than 4% being monomeric. Analysis by non-reducing SDS-PAGE and immunoblotting show that the beta chains expressed alone are in Triton X-100 insoluble, disulfide-linked aggregates. Co-expression of both subunits in insect cells results in only a small fraction (less than 15%) of the alpha chains being assembled as the active recombinant enzyme, with at least 22% of the active recombinant enzyme localized to the plasma membrane as determined by a biochemical assay. The small amount of beta chain at the plasma membrane in cells that express both subunits is beyond the limit of detection by the biochemical assay. Immunoprecipitation of Triton X-100 soluble alpha chains from radio-labeled cells expressing both subunits shows that the alpha chains are mostly in large aggregates containing beta chains. These results suggest that, in insect cells, the availability of correctly folded beta chains is the rate limiting step in the assembly of active Na+/K+ ATPase.
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PMID:Biochemical characterization of the subunits of the Na+/K+ ATPase expressed in insect cells. 936 64

Sucrose density gradient purified plasma membranes isolated from brown adipose tissue of cold-acclimated hamsters (4-10 weeks at 0-4 degreesC) were analysed for the content of the short (GsalphaS) and long (GsalphaL) variants of Gsalpha protein (the alpha subunit of the stimulatory G protein) and compared with the membranes isolated from control animals. The relative ratio between the two variants (GsalphaS/GsalphaL) decreased from 0.48 to 0.24 (P<0.01). This result, obtained by electrophoretic resolution of membrane proteins by standard SDS-PAGE and an immunoblot analysis with an antiserum oriented against an internal sequence of Gsalpha, was verified by resolution on urea-containing gels and an antiserum oriented against the C-terminus decapeptide of Gsalpha. Under these conditions, the GsalphaS/GsalphaL ratio was decreased from 0.41 to 0.31 (P<0.05). The total amount of both isoforms (GsalphaS plus GsalphaL) decreased to 83% (P<0.05) or 68% (P<0.01) by standard or urea SDS-PAGE respectively. These data demonstrate that cold-acclimation of hamster brown adipose tissue is associated with preferential decrease in the plasma membrane density of the short variant of the Gsalpha protein.%This decrease was paralleled by an increase in the other plasma membrane constituents, [3H]CGP12177 binding sites, [3H]ouabain binding sites and Na,K-ATPase activity to 147%, 212% and 191% respectively.
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PMID:The decrease in the short variant of gsalpha protein is associated with an increase in [3H]CGP12177 binding, [3H]ouabain binding and Na, K-ATPase activity in brown adipose tissue plasma membranes of cold-acclimated hamsters. 992 80

Through reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers, a VCP homolog was identified in African trypanosomes. Sequence analysis shows a 72 and 64% deduced amino acid identity, respectively, with mouse VCP and yeast Cdc48p. Southern analysis indicates tbVCP to have a single locus with two alleles. Antibodies generated against recombinant protein recognize a 95 kDa protein in whole cell lysates of both procyclic and bloodstream trypanosomes. There is an approximately four-fold greater expression of TbVCP protein in the procyclic stage of the trypanosome life cycle. Subcellular fractionation and immunofluorescence with anti-TbVCP antibodies indicate the majority of TbVCP to be cytoplasmically localized with a small subset associated with membranes. Sucrose velocity sedimentation and gel filtration size analysis studies suggest that TbVCP is a homohexameric particle as has been demonstrated with other VCP homologs. Also like other VCP homologs, TbVCP contains an NEM-inhibitable ATPase activity. This is the first characterization of an AAA (ATPases Associated with a variety of cellular Activities) family member in African trypanosomes.
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PMID:Molecular cloning and biochemical characterization of a VCP homolog in African trypanosomes. 1002 5

Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H+-ATPase to bafilomycin-A and NO3-. Inhibition of the H+-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H+-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism.
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PMID:ATP-induced sucrose efflux from red-beet tonoplast vesicles. 1092 6

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.
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PMID:Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii. 1100 72

To investigate the characteristic features of diabetic neuropathy in type 2 diabetes mellitus, Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of human type 2 diabetes mellitus, and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats were fed with or without sucrose and/or an aldose reductase inhibitor, [5-(3-thienyl) tetrazol-1-yl] acetic acid (TAT), for 24 weeks, and physiological, biochemical and morphological assessments were performed. Sucrose administration caused remarkable hyperglycemia in OLETF rats but not in LETO rats. Sucrose-fed OLETF rats demonstrated delayed nerve conduction velocity, decreased coefficient of variation of R-R interval, reduced sciatic nerve blood flow, increased platelet aggregation activity, a lower concentration of erythrocyte 2,3-diphosphoglycerate, and decreased Na+/K+-ATPase activity in sciatic nerves, compared with the non-sucrose-fed OLETF and LETO rats. TAT prevented all these deficits except hyperglycemia. Sorbitol and fructose accumulation and myo-inositol depletion in tail nerves of sucrose-fed OLETF rats were ameliorated by TAT. Myelinated fiber size and density in sural nerves of sucrose-fed OLETF rats were decreased and increased, respectively, compared with non-sucrose-fed OLETF and LETO rats. These morphological abnormalities were normalized by TAT. These observations suggest that the sucrose-fed OLETF rat developed diabetic neuropathy not only electrophysiologically but also histologically, and that an aldose reductase inhibitor, TAT, possesses therapeutic value for the treatment of diabetic neuropathy.
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PMID:Physiological and morphometric analyses of neuropathy in sucrose-fed OLETF rats. 1113 77

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.
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PMID:Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters. 1126 Apr 67

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.
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PMID:Nitrophenylphosphate as a tool to characterize gill Na(+), K(+)-ATPase activity in hyperregulating Crustacea. 1169 3


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