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)

The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys. By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation. H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored. Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased. Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable. Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.
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PMID:The gamma subunit of the Escherichia coli ATP synthase. Mutations in the carboxyl-terminal region restore energy coupling to the amino-terminal mutant gamma Met-23-->Lys. 841 64

Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.
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PMID:Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function. 855 May 67

We present the nucleotide sequence of a 5207-bp-long region of the mitochondrial genome of the dermatophyte Trichophyton rubrum. This represents about 1/5th of the total genome and extends a previous study. From the 5' end of the present sequence, the order of genes is as follows: the end of the ND4 gene, the gene coding for subunit 6 of ATPase, the gene coding for the small ribosomal RNA (SSU rRNA), the tyrosyl tRNA gene, the ND6 gene, the COXIII gene, the ATPase 8 subunit gene and a cluster of tRNAs genes corresponding respectively to the lysine, glutamine, asparagine, isoleucine and tryptophan isoacceptors. The interesting features of this region are its compact organisation, the presence of subunit 8 of the ATPase gene and the secondary structure of SSU rRNA which is close to that of Aspergillus nidulans. On the basis of the order of the genes, which is essentially similar to that of A. nidulans, we can also assume that the LSU rRNA subunit gene should be just upstream of this sequenced region.
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PMID:Organisation of the mitochondrial genome of Trichophyton rubrum. DNA sequence analysis of the ND4 gene, the ATPase subunit-6 gene, the ribosomal RNA small-subunit gene, the ND6 gene, the COXIII gene, the ATPase subunit-8 gene and six tRNA genes that correspond respectively to the tyrosine, lysine, glutamine, asparagine, isoleucine and tryptophan isoacceptors. 859 86

Site-directed mutagenesis studies identifying residues important to energy transduction in the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. Mutations blocking the crucial E1P to E2P transition are located in the small and the large cytoplasmic domains, in the stalk segment S4 linking transmembrane segment M4 with the catalytic site, as well as in transmembrane segments M4 and M8. Mutations that block the dephosphorylation of the E2P phosphoenzyme intermediate are located in transmembrane segments M4, M5, and M6, i.e., in the same domain as the Ca(2+)-binding sites. Removal of the sidechain of Tyr763 located at the boundary between transmembrane segment M5 and the corresponding stalk segment S5 linking M5 with the catalytic site leads to uncoupling of ATP hydrolysis from Ca2+ uptake. Uncoupling may be due to efflux through the Ca(2+)-ATPase of Ca2+ that has been transported, and may thus be caused by a defective gating process in the late part of the catalytic cycle. A nearby located residue Lys758 is also involved in energy coupling, since its substitution with Ile activates dephosphorylation at high pH and slows the E2 to E1 transition.
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PMID:Site-directed mutagenesis studies of energy coupling in the sarcoplasmic reticulum Ca(2+)-ATPase. 868 43

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.
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PMID:Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme. 872 Jan 51

1. Relaxation of airway smooth muscle induced by vasoactive intestinal peptide (VIP) is mediated by adenosine 3':5' cyclic monophosphate (cyclic AMP). An interaction between the synthesis of cyclic AMP and enzymic activity of the plasmalemmal sodium pump (Na(+)-K(+)-ATPase) exists in certain isolated cell systems. This study sought to determine the contribution of Na(+)-K(+)-ATPase activity to relaxation of airway smooth muscle evoked by VIP. 2. All experiments were performed on isolated strips of guinea-pig trachea from which the epithelium had been removed. VIP was a more potent relaxant in tissues that were contracted with carbachol than those contracted with an equi-effective depolarizing concentration of K+. 3. Ouabain (0.1 microM-10 microM) induced contraction of tracheal strips. Contraction to ouabain (5 microM) was abolished following incubation of tissues with K(+)-free, or Ca(2+)-free (+ EGTA, 0.1 mM) physiological solutions. The contractile response to ouabain (5 microM) was not influenced significantly by exposure of the tissues to atropine (1 microM), phentolamine (5 microM) and diphenhydramine (1 microM) for 60 min. 4. Tissues were incubated with ouabain (5 microM; 60 min) or K(+)-free physiological solution (60 min) to inhibit Na(+)-K(+)-ATPase activity. These procedures reduced relaxation induced by VIP, peptide histidine isoleucine, forskolin, isoprenaline and sodium nitroprusside. 5. Relaxation to VIP was impaired significantly following exposure of tissues to a low Na+ solution (30 min) or amiloride (500 microM; 30 min). 6. Ouabain-sensitive uptake of 86Rb was measured in tracheal strips (devoid of epithelium and cartilage) as an index of Na(+)-K(+)-ATPase activity. VIP (1 microM; 2 min) caused a 4.7 fold stimulation of ouabain-sensitive uptake of 86Rb. This effect was impaired significantly by low Na+ solution. 7. The results suggest that (i) relaxation of tracheal smooth muscle to VIP is sensitive to procedures that inhibit activity of Na(+)-K(+)-ATPase and invoke a role for altered sodium pump function in the mechanisms that underlie cyclic AMP-dependent relaxation; and (ii) VIP stimulates ouabain-sensitive uptake of 86Rb in airway smooth muscle in a Na(+)-dependent manner.
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PMID:Stimulation of sodium pump by vasoactive intestinal peptide in guinea-pig isolated trachea: potential contribution to mechanisms underlying relaxation of smooth muscle. 876 78

The peptide Ac-MEKVQYLTRSAIRRASTIEMPQQAR (Ac-PLB(1-25)) representing residues 1-25 of phospholamban (PLB) inhibited the maximal activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum by about 53%, with a Kd value of 5 microM; the equivalent non-acetylated peptide PLB(1-25) had no effect. However, it was found that the non-acetylated peptide increased the effective Kd value for inhibition by Ac-PLB(1-25) consistent with competitive binding to the ATPase, with a Kd value of 8 microM for PLB(1-25). The non-acetylated peptide must therefore be able to bind to the ATPase, but in a conformation that does not lead to inhibition of the ATPase. The identity of the N-terminal residue is important in determining the strength of binding; replacement of the Met residue by Ile led to fourfold weaker binding, again with only binding of the acetylated peptide leading to inhibition of ATPase activity.
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PMID:The effect of N-terminal acetylation on Ca(2+)-ATPase inhibition by phospholamban. 880 39

We tested the hypothesis that nutritional state affects seawater acclimation by transferring either fed or food-deprived (2 weeks) male tilapia (Oreochromis mossambicus) from fresh water to full-strength sea water. Food-deprivation resulted in a significant increase in plasma concentrations of Na+, Cl-, cortisol, glucose, total amino acid, glutamate, serine and alanine, and in hepatic pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities, whereas the prolactin-188 to prolactin-177 ratio (tPRL188:tPRL177) and plasma prolactin-188 (tPRL188), lactate, arginine and hepatic glycogen content and hepatic alanine aminotransferase (AlaAT) and 3-hydroxyacyl-Coenzyme A dehydrogenase (HOAD) activities were lower than in the fed group. Seawater transfer significantly increased the tPRL188:tPRL177 ratio and plasma concentrations of Na+, Cl-, K+, growth hormone (GH), glucose, aspartate, tyrosine, alanine, methionine, phenylalanine, leucine, isoleucine and valine levels as well as gill Na+/K+-ATPase activity and hepatic PK and LDH activities, whereas plasma tPRL177, tPRL188, glycine and lysine concentrations were significantly lower than in fish retained in fresh water. There was a significant interaction between nutritional state and salinity that affected the tPRL188:tPRL177 ratio and plasma concentrations of Cl-, GH, glucose, aspartate, tyrosine, serine, alanine, glycine, arginine and hepatic PK, LDH, AlaAT, aspartate aminotransferase, glutamate dehydrogenase and HOAD activities. These results, taken together, indicate that food-deprived fish did not regulate their plasma Cl- levels, despite an enhancement of plasma hormonal and metabolic responses in sea water. Our study also suggests the possibility that plasma prolactin and essential amino acids may be playing an important role in the seawater acclimation process in tilapia.
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PMID:Food-deprivation affects seawater acclimation in tilapia: hormonal and metabolic changes 932 Mar 94

The highly conserved lysine residue Lys758 in the fifth stalk segment of the sarcoplasmic reticulum Ca2+-ATPase was substituted with either isoleucine or arginine by site-directed mutagenesis. The substitution with arginine was without significant effects on Ca2+-ATPase function, whereas multiple changes of functional characteristics were observed with the Lys758 --> Ile mutant. These included insensitivity of ATPase activity to the calcium ionophore A23187, an alkaline shift of the pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinities for Ca2+ and inorganic phosphate, as well as increased sensitivity to inhibition by vanadate. Analysis of the partial reaction steps of the enzyme cycle traced these changes to two steps. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) was increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the dephosphoenzyme with low Ca2+ affinity (E2) to the Ca2+-bound form activated for phosphorylation (E1Ca2) was reduced in the mutant, and the ATP-induced rate enhancement of this step required higher ATP concentrations in the mutant compared with the wild type.
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PMID:Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition. 937 9

The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem. 269, 26598-26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry 36, 11865-11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+ transport. A single mutation in the S3 stalk segment (Gly257 --> Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor.
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PMID:Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition. 958 34


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