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)

Small samples of the masseter and temporalis muscles of six adult male cats were dissected free, snap-frozen and cryosectioned. Serial sections from areas with muscle spindles were histochemically stained for myofibrillar ATPase after incubation in acid and alkali buffer, and for reactivity to two different myosin isoform-specific antibodies. Differential reactivity to these four stains identified bag1, bag2 and chain intrafusal fibre types. The most typical distribution of fibres within the spindle was 1 bag1, 1 bag2 and 3-4 chains. Although a few spindles were found in the anterior area of the temporalis that attaches to the coronoid process of the mandible and in the anterior area of the superficial masseter, the vast majority were found in the deepest portion of the masseter. Here spindles were characteristically with a number of simple spindles fused together into complex units.
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PMID:Fibre type distribution in the muscle spindles of cat jaw-elevator muscles. 821 92

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.
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PMID:In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants. 829 80

A second isoform of the Artemia franciscana sarcoplasmic or endoplasmic reticulum Ca-ATPase has been identified through the isolation of cDNA clones. This isoform differs from the previously identified one only at the C-terminal end of the protein. The last 6 amino acids of the former isoform change to 30 hydrophobic amino acids in the newly identified isoform that have the potentiality of being an additional transmembrane domain. The two A. franciscana isoforms are highly homologous to the two isoforms coded by the mammalian and bird SERCA2 gene, except that the C-terminal extensions share their hydrophobic character but have no significant amino acid homology. The isolation of genomic clones coding for this region of the gene shows that both isoforms arise from the same gene by alternative splicing. The donor splicing site of the penultimate exon can either be recognized and fused to the last exon, giving rise to the mRNA coding for the shortest protein, or remain unrecognized, in which case a polyadenylation site is recognized before the last exon of the gene and the mRNA coding for the largest protein is originated. The alternative splicing used to originate the two isoforms is similar in Artemia and vertebrates. The conservation of the alternative splicing between species so distant in evolution suggests an important physiological role for the existence of the two isoforms of the protein.
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PMID:Similar alternative splicing events generate two sarcoplasmic or endoplasmic reticulum Ca-ATPase isoforms in the crustacean Artemia franciscana and in vertebrates. 831 76

The gene encoding proglucagon is expressed in the A-cells of the endocrine pancreas and the L-cells of the gastrointestinal tract. Proglucagon-derived peptides have also been detected in different regions of the central nervous system; however, proglucagon mRNA transcripts have been localized predominantly to the brainstem and hypothalamus. We have studied proglucagon gene expression in the brain of mice using the reverse transcription-polymerase chain reaction technique. Proglucagon mRNA transcripts were detected in the cortex, cerebellum, and brainstem of embryonic day 19 mouse brain and in the cortex, cerebellum, brainstem, and hypothalamus of 2- to 12-week-old wild-type mice. Age-specific changes in proglucagon gene expression were observed, with a progressive decrease in the levels of proglucagon mRNA transcripts detected in the cortex of older mice. In contrast, a progressive increase in the levels of proglucagon mRNA transcripts was detected with increasing age in the brainstem. Proglucagon gene expression was also examined in the brain of glucagon-simian virus-40 (SV40) T-antigen transgenic mice. The levels of proglucagon gene expression in transgenic cerebellum and brainstem were relatively greater than those in wild-type mice on embryonic day 19, and increased levels of proglucagon mRNA transcripts were observed in transgenic cerebellum, brainstem, and hypothalamus at 2 weeks. In contrast, the levels of proglucagon mRNA transcripts were markedly reduced in the brain of 5-week-old transgenic mice. A reduction in the levels of SV40 T-antigen mRNA transcripts was also detected in the brain of transgenic mice at 5 weeks, but no suppression of the mRNA transcripts for the neuropeptide somatostatin was observed in 5-week-old transgenic brain. The results of these studies suggest that proglucagon mRNA transcripts are more widely distributed in the mouse central nervous system than previously demonstrated and that proglucagon gene expression in the brain appears to be regulated in a region- and age-specific manner. The coordinate region-specific expression and regulation of mRNA transcripts derived from the endogenous mouse proglucagon gene and a glucagon-SV40 T antigen transgene in the central nervous system of mice harboring a transgene containing 2.0 kilobases of rat proglucagon gene 5'-flanking sequences fused to the coding sequence of SV-4 large T-antigen suggest that neural-specific elements residing within the first 2.0 kilobases of glucagon gene 5'-flanking sequences are sufficient for the correct targeting and regulation of proglucagon gene expression in the brain.
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PMID:Region- and age-specific differences in proglucagon gene expression in the central nervous system of wild-type and glucagon-simian virus-40 T-antigen transgenic mice. 831 63

The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
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PMID:ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. 836 61

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.
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PMID:Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae. 838 51

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cl- channels of the gastric parietal cell that are active at low pH. 839 3

The two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli. The GST-L peptide contained the hydrophilic region from Ala340 to Ser660. The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E. coli culture. The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL. ATP competitively displaced the TNP-ATP binding. The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein. The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide. Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5.
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PMID:Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma membrane H(+)-ATPase. 840 44

The ATPase inhibitor protein of the rat liver mitochondrial ATP synthase/ATPase complex has been cloned from a rat liver cDNA library, and its nucleotide sequence determined. The sequence is highly homologous to both the bovine heart (approximately 70%) and the yeast inhibitor proteins (approximately 40%). The deduced protein sequence is 107 amino acids in length, and based on homology to the bovine heart protein, the first 25 N-terminal amino acids encode a putative mitochondrial targeting sequence. The "mature" protein (without the targeting sequence) fused to the maltose binding protein has been overexpressed in Escherichia coli. The maltose binding protein was used as a handle for the development of a rapid one-step purification of the fusion protein by affinity chromatography on an amylose resin. The purified fusion protein was cleaved with Factor Xa protease at the fusion junction, and the resulting ATPase inhibitor protein was purified to > 90% purity. The purified, overexpressed inhibitor protein displays normal inhibitor activity. The protein inhibits ATP hydrolysis catalyzed by the ATP synthase/ATPase complex in submitochondrial particles in a manner kinetically indistinguishable from the same protein purified from rat liver mitochondria, and exhibits a specific activity of approximately 10,000 units/mg. The secondary structure of the inhibitor protein was determined by circular dichroism spectropolarimetry. The experimentally determined structure shows a high content of alpha-helix and is in good agreement with sequence-based structural predictions. As the function of the inhibitor protein is known to exhibit a high dependence on pH, a study of the pH dependence of inhibitor secondary structure was performed. It is shown that as pH is lowered, conditions which activate inhibitory capacity, the protein loses significant alpha-helical structure. This is the first report of the overexpression in E. coli of a functional ATPase inhibitor protein. Secondary structural analysis of this protein indicates that conversion from its active to its inactive form involves a significant conformational change.
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PMID:Regulation of the mitochondrial ATP synthase/ATPase complex: cDNA cloning, sequence, overexpression, and secondary structural characterization of a functional protein inhibitor. 844 67

The bcl-2 gene can potentially encode 26- and 22-kDa proteins that differ only in their carboxyl tails because of an alternative splicing mechanism. The larger of these proteins contains a hydrophobic transmembrane domain within its carboxyl terminus, resides (at least in part) in mitochondrial membranes and has been shown to prolong cell survival by blocking programmed cell death (also termed "apoptosis"). To explore the function of the shorter 22-kDa Bcl-2 protein that lacks a transmembrane domain, DNAs encoding p26-Bcl-2-alpha or p22-Bcl-2-beta were expressed in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D. In contrast to p26-Bcl-2 alpha that markedly prolonged cell survival, p22-Bcl-2-beta did not extend the survival of 32D cells when cultured in the absence of IL-3. Expression in 32D cells of a chimeric DNA that fused portions of the open reading frame common to Bcl-2-alpha and Bcl-2-beta (amino-acids 1-195) with sequences encoding the transmembrane and cytosolic domains of the IL-2 receptor-alpha protein resulted in production of a Bcl-2/IL-2R fusion protein that was capable of prolonging 32D cell survival in the setting of IL-3 withdrawal. Based on fractionation of cells to produce crude heavy membrane, light membrane, nuclei, and cytosolic preparations, much of the p22-Bcl-2-beta protein appeared to reside in the cytosol, whereas Bcl-2-alpha and the Bcl-2/IL-2R chimeric proteins were found exclusively in fractions that also contained the inner mitochondrial membrane protein F1-beta-ATPase. Taken together, these findings demonstrate the importance of membrane association for the function and intracellular targeting of the apoptosis-blocking Bcl-2 protein. Furthermore, despite the strong evolutionary conservation of the carboxyl regions of Bcl-2-alpha proteins observed previously for mammalian and avian species, these data suggest that a heterologous transmembrane domain can be substituted without loss of function.
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PMID:Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival. 849 57


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