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)

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.
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PMID:The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity. 132 2

The presence of an H+/K(+)-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K(+)-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K(+)-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pHi, measured in HCO(3-)-free medium, was 7.62 +/- 0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40 +/- 0.03 (n = 18) after 35 days in culture. Recovery of pHi following a NH+4/NH3 pulse could be reduced by either 100 microM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K(+)-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.
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PMID:The colon carcinoma cell line Caco-2 contains an H+/K(+)-ATPase that contributes to intracellular pH regulation. 133 76

The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.
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PMID:Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP. 133 71

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.
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PMID:Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. 133 46

We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.
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PMID:Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. 134 4

We have investigated the role, number, and identity of glutamate (or aspartate) residues involved in cation occlusion on Na+, K(+)-ATPase, using the carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD). Extensive use is made of selectively trypsinized Na+,K(+)-ATPase--the so-called "19-kDa membranes"--containing a 19-kDa COOH-terminal, smaller (8-11 kDa) membrane-embedded fragments of the alpha chain, and a largely intact beta chain; these membranes have normal Rb+ and Na+ occlusion capacities. The 19-kDa peptide and a smaller (approximately 9 kDa) unidentified peptide(s) are labeled by [14C]DCCD in a Rb(+)-protectable fashion. Rb(+)-protected [14C]DCCD incorporation into the "19 kDa membranes" and into native Na+,K(+)-ATPase is linearly correlated with inactivation of Rb+ occlusion. Similar linear correlations are observed when Rb(+)-protected [14C]DCCD incorporation is measured by examination of labeling of 19-kDa peptide purified from "19-kDa membranes" or of alpha chain purified from native enzyme. Stoichiometries, estimated by extrapolation, are as follows: (for "19-kDa membranes") close to one DCCD per Rb+ site and one DCCD per 19-kDa peptide; and (for native enzyme) close to two DCCD per phosphoenzyme and two DCCD per alpha chain. We suggest that each of two K+ (or Na+) sites contains a carboxyl group, one located in the 19-kDa peptide and one elsewhere in the alpha chain. After cyanogen bromide digestion of purified, labeled alpha chain, or of 19-kDa peptide, a labeled fragment of apparent M(r) approximately 4 kDa was detected and was identified as that with NH2-terminal Lys-943. Rb(+)-protected [14C]DCCD incorporation was associated almost exclusively with Glu-953. We suggest that the cation occlusion "cage" consists of ligating groups donated by different trans-membrane segments and includes two carboxyl groups such as Glu-953 (and perhaps Glu-327) as well as neutral groups, in two K+ (or Na+) sites, but only neutral groups in the third Na+ site.
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PMID:Chemical modification of Glu-953 of the alpha chain of Na+,K(+)-ATPase associated with inactivation of cation occlusion. 135 83

The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane.
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PMID:Local anesthetics induce fast Ca2+ efflux through a nonenergized state of the sarcoplasmic reticulum Ca(2+)-ATPase. 137 93

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine. 138 24

cDNA clones encoding the 70,000 relative molecular weight (M(r)) subunit of the bovine vacuolar proton-adenosine triphosphatase (H(+)-ATPase) were isolated, and a total length of 3.1 kb in overlapping clones from bovine kidney and brain libraries was sequenced. The cDNA contains a 1.9-kb coding region and yields a deduced protein sequence of 618 amino acids. The subunit sequence has 50% amino acid identity with the corresponding subunit from yeast, carrot, and Neurospora vacuolar H(+)-ATPases. The internal regions of the protein are most highly conserved, whereas the NH2- and COOH-terminals exhibit variability. mRNA levels of the M(r) 70,000 subunit were examined in multiple bovine tissues and were found to be expressed at highest levels in kidney medulla and cortex, at moderate levels in brain and adrenal gland, and at low levels in liver, muscle, and heart.
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PMID:cDNA sequence and tissue expression of bovine vacuolar H(+)-ATPase M(r) 70,000 subunit. 138 91

A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15,807 Da (pI = 9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H(+)-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.
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PMID:Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of vacuolar H(+)-ATPases: organellar and tissue distribution of 16 kDa proteolipids. 140 Feb 63


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