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)

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.
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PMID:Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454. 753 69

The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.
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PMID:Beta-gamma subunit interaction is required for catalysis by H(+)-ATPase (ATP synthase). Beta subunit amino acid replacements suppress a gamma subunit mutation having a long unrelated carboxyl terminus. 755 18

Regulation of the ATPase activity of smooth and nonmuscle myosin II involves reversible phosphorylation of the regulatory light chain (RLC). The RLC from skeletal muscle myosin (skRLC) is unable to confer regulation (myosin is locked in an inactive state) to smooth muscle myosin when substituted for the endogenous smooth RLC (smRLC). Studies of chimeric light chains comprised of the N- or C-terminal half of each skRLC and smRLC suggest that the structural basis for the loss of this regulation is within the C-terminal half of the RLC (Trybus, K.M., and Chatman, T.A. (1993) J. Biol. Chem. 268, 4412-4419). The purpose of this study is to delineate the structural elements within the C-terminal half of the smRLC that are absent in the skRLC and are necessary for regulation. By sequence comparison, six residues, Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg-160, which are conserved in regulated myosin RLCs but missing in nonregulated myosin RLCs, were identified in smRLC. To test whether these amino acids provide the missing structural elements necessary for phosphorylation-mediated regulation, a skRLC was engineered that replaced the corresponding skRLC amino acids (positions 100, 120, 126, 127, 140, and 157, respectively) with their smRLC counterparts. Using a newly developed RLC exchange procedure, the purified mutant protein was evaluated for its ability to regulate chicken gizzard smooth muscle myosin. Substitution of the six conserved amino acids into the skRLC completely restored phosphorylation-mediated regulation. Thus, a subset of these amino acids, including four basic arginine residues located in the E, F, G, and H helices which are missing in skRLC, may be the structural coordinates for the phosphorylserine in the N terminus. Based on this result, the regulation of glycogen phosphorylase is discussed as a model for the regulation of smooth muscle myosin.
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PMID:Restoration of phosphorylation-dependent regulation to the skeletal muscle myosin regulatory light chain. 755 73

The protease subtilisin has been reported to cleave skeletal muscle G-actin between Met 47 and Gly 48 generating a core fragment of 33 kDa and a small N-terminal peptide, which remains attached to the core fragment [Schwyter, D. Phillips, M., & Reisler, E. (1989) Biochemistry 28, 5889-5895]. However, amino acid sequencing and mass spectroscopy of subtilisin cleaved-actin revealed two cleavage sites, one between Met 47 and Gly 48 and a second between Gly 42 and Val 43, generating an actin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we describe a procedure for purifying the actin core fragment and the attached N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromatography. After removal of the pentapeptide, the salt-induced polymerization of actin was abolished. However, the purified fragments could be polymerized by addition of salt plus myosin subfragment 1 or salt plus phalloidin as shown by sedimentation and fluorescence increase using N-(1-pyrenyl)iodoacetamide labeled actin. These results confirm earlier reports proposing that cleavage in the DNase I binding loop is affecting the ion induced polymerization of actin [Higashi-Fujime, S., et al. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subactin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct binding of subactin to DNase I was verified by gel filtration and to myosin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.
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PMID:Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop. 757 93

The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.
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PMID:Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon. 765 12

The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone.
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PMID:A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. 765 24

The asymmetry of Escherichia coli F1-ATPase (ECF1) has been explored in chemical modification experiments involving two mutant enzyme preparations. One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant beta G149C:V198A). The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the epsilon subunit (mutant beta G149C:V198A:E381C/epsilon S108C). On CuCl2 treatment of this second mutant, there is cross-linking of one copy of the beta subunit to gamma via the Cys at 381, a second to the epsilon subunit (between beta Cys381 and epsilon Cys108), while the third beta subunit in the ECF1 complex is mostly free (some cross-linking to delta); thereby distinguishing the three beta subunits as beta gamma, beta epsilon, and beta free, respectively. Both mutants have ATPase activities similar to wild-type enzyme. Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys149, dicyclohexyl carbodiimide (DCCD) which reacts with Glu192, and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr297. Thus, beta gamma reacted with DCCD but not NEM or NbfCl; beta free was reactive with all three reagents; beta epsilon reacted with NEM, but was poorly reactive to DCCD or NbfCl. There was a strong nucleotide dependence of the reaction of Cys149 in beta epsilon (but not in beta free) with NEM, indicative of the important role that the epsilon subunit plays in functioning of the enzyme.
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PMID:Asymmetry of Escherichia coli F1-ATPase as a function of the interaction of alpha-beta subunit pairs with the gamma and epsilon subunits. 765 34

The binding of monoclonal antibody M7-PB-E9 to the alpha-subunit of Na+,K(+)-ATPase partially inhibits enzyme activity (35%) in competition with ATP, while in the presence of magnesium it stimulates the rate of ouabain binding severalfold [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. These effects have been shown to result from an antibody-induced shifting of the enzyme's E1 <==> E2 conformational equilibrium to the right that affects all enzyme-ligand interactions except that with Mg2+ [Abbott, A.J., & Ball, W.J. (1992) Biochemistry 31, 11236-11243]. In order to identify the location of the M7-PB-E9 epitope, proteolytic fragments of the lamb kidney enzyme were generated and the immunoreactive alpha fragments were identified by Western blot analyses. These studies revealed a 47-kDa tryptic fragment, which bound both M7-PB-E9 and a -COOH terminus specific antisera and NH2-terminal sequencing showed to originate at Ala-590. Digestion with Staphylococcus aureus V8 protease produced a 36-kDa -COOH-terminus fragment which originated at Gly-697 and did not contain the antibody epitope. Thus the intracellular sequence region Ala-590 to Gly-697 was shown to contain the antibody epitope. When M7-PB-E9's ability to recognize the alpha subunits from various species and tissues was determined and correlated with available sequencing data, only Ser-646 was present in the highly reactive lamb, pig, and avian kidney alpha 1 proteins and altered (Asn) in the poorly recognized Xenopus and rat kidney and Torpedo electroplax organ enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The epitope for the inhibitory antibody M7-PB-E9 contains Ser-646 and Asp-652 of the sheep Na+,K(+)-ATPase alpha-subunit. 768 92

Biologically active amidated gastrin is synthesized by carboxyl-terminal alpha-amidation of a glycine-extended progastrin post-translational processing intermediate (G-Gly). Although plasma levels of G-Gly are equivalent to those of gastrin, G-Gly has essentially no acute effect on gastric acid secretion. However, we have observed that inhibition of gastrin amidation leads to increased plasma concentrations of G-Gly and enhanced gastric acid secretion. We hypothesized, therefore, that G-Gly might have a chronic effect to increase H+,K(+)-ATPase expression in gastric parietal cells. In the present studies, we observed that a 2-day preincubation with G-Gly significantly enhanced histamine-stimulated [14C]aminopyrine uptake by isolated canine gastric parietal cells but acutely administered G-Gly had no effect. On Northern blot analysis, both G-Gly and gastrin dose-dependently increased H+,K(+)-ATPase alpha-subunit gene expression with maximal induction (225 +/- 35 and 170 +/- 29% of basal, mean +/- S.E.) achieved at concentrations of 10(-9) M G-Gly and 10(-8) M gastrin, respectively. Using an H+,K(+)-ATPase alpha-subunit gene-luciferase chimeric reporter construct transfected into primary cultured parietal cells, we observed that both G-Gly and gastrin increased luciferase activity in a manner similar to that obtained by Northern blot analysis. L365,260, a specific gastrin/CCKB receptor antagonist, completely reversed the stimulation of luciferase activity induced by gastrin but had no effect on G-Gly-stimulated activity. Gastrin increased [Ca2+]i, although G-Gly did not, however, genistein (a tyrosine kinase inhibitor) significantly reduced induction of luciferase activity by both G-Gly and gastrin. Specific binding of 125I-Leu15-G2-17-Gly to gastric parietal cells was dose-dependently displaced by G2-17-Gly but not by gastrin nor L365,260. Gastrin peptides truncated at the carboxyl- (G1-13) and amino terminus (G5-17-Gly) both induced H+,K(+)-ATPase alpha-subunit gene expression and inhibited 125I-Leu15-G2-17-Gly binding, but were less potent than G2-17-Gly. These data indicate that G-Gly may have a functional role in potentiating gastric acid secretagogue action via enhanced expression of the gene responsible for H+ generation through action at a novel receptor that can be distinguished from the gastrin/CCKB receptor. Thus, both the substrate and product of the terminal progastrin processing reaction appear to have complementary functions in regulation of gastric acid secretion.
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PMID:Glycine-extended progastrin processing intermediates induce H+,K(+)-ATPase alpha-subunit gene expression through a novel receptor. 774 46

The Ser14 hydroxyl group of actin is one of six groups that potentially form hydrogen bonds with the gamma-phosphate of the ATP bound in the cleft separating the two domains of the protein. To understand the importance of this group in actin function, we mutated Ser14 of Saccharomyces cerevisiae actin and studied the effects of these mutations in vivo and in vitro. Substitution of Cys of Gly resulted in cell death. Substitution of Thr for Ser resulted in an actin with wild-type properties in vivo and in vitro. Cells carrying the Ser14-->Ala (S14A) mutation were viable but displayed a temperature sensitive lethality at 37 degrees C preceded by delocalization of actin patches, the appearance of bar-like structures, and finally the disappearance of identifiable actin structures. The mutation caused no effect on the critical concentration of polymerization but resulted in an actin with an increased rate of polymerization, an altered protease susceptibility, and a decreased filament ATPase activity. At 37 degrees C, Mg-, but not Ca-S14A-actin irreversibly lost the ability to polymerize. These results demonstrate the importance of the ATP-Ser14 hydroxyl hydrogen bond in regulating actin function in vivo and in vitro and the magnification of the effects of the mutation when Mg2+ is substituted for Ca2+ in the protein.
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PMID:A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro. 774 77


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