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

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Chemical modification of the Ca(2+)-ATPase with phenylglyoxal, as a modifier of arginine residues, leads to an almost total loss of the ATPase activity. The presence of nucleotides in the reaction medium protects against the binding of 18 nmol of phenylglyoxal/mg of protein and this reduction in the binding of phenylglyoxal is accompanied by a substantial retention of ATPase activity. The incorporation of phenylglyoxal to the protein alters neither calcium binding nor phosphorylation from inorganic phosphate. Nevertheless the binding of nucleotides is dramatically inhibited and, consequently, so is phosphorylation from ATP. Fluorescein 5'-isothiocyanate labelling of the phenylglyoxal-modified ATPase is not affected but, on the other hand, phenylglyoxal is not able to modify the fluorescein 5'-isothiocyanate-prelabelled ATPase. The way in which ATPase inhibition depends on the presence of phenylglyoxal indicates that this process occurs in a pseudo-first-order reaction. However, the dependence of the apparent first-order rate constant on phenylglyoxal concentration appears to be more complex and an inhibition mechanism of two steps, with phenylglyoxal binding, has to be taken into account. 2. We have found that phenylglyoxal labels both A and B tryptic fragments, but only B fragment labelling is prevented by ATP. The sequencing of peptides from mild acid hydrolysis of phenylglyoxal-labelled ATPase shows that phenylglyoxal is located in the Ala506-Gly595 peptide that is a part of the B fragment. 3. We conclude that phenylglyoxal inactivates the calcium pump in a two-step mechanism in which the second step is irreversible. Phenylglyoxal labels an arginyl residue in the Ala506-Gly595 peptide that can be protected by the binding of ATP to its site.
...
PMID:Involvement of an arginyl residue in the nucleotide-binding site of Ca(2+)-ATPase from sarcoplasmic reticulum as seen by reaction with phenylglyoxal. 876 69

A simple and rapid protein chemical approach for determining the transmembrane structure of membrane proteins is described. The method involves single substitutions of consecutive amino acid residues, within putative transmembrane segments, to cysteine. This is followed by the analysis of their susceptibility to modification by maleimides with different physico-chemical properties. Fluorescein-5-maleimide (FM), being hydrophilic, modified only residues located in the aqueous environment, while the hydrophobic reagent, benzophenone-4-maleimide (BM) modified residues exposed to the lipid phase. These probes are large enough to cause an increase in the molecular weight of relatively small membrane proteins or polypeptide fragments, which is detectable by SDS-PAGE. Modification by much smaller probes, such as N-ethylmaleimide (NEM), could also be monitored indirectly by the ability to prevent SDS-solubilized protein from being modified with fluorescein-5-maleimide. The approach is demonstrated with the proteolipid complex of the vacuolar H(+)-ATPase expressed in yeast and with the putative Isk K(+)-channel expressed and radiolabelled in E. coli. The advantages of this approach are: (1)it is rapid, easy and inexpensive, (2) detection of the modification of engineered cysteines is simple, (3) it requires only minute quantities of the protein, (4) the protein does not require purification, (5) a broad range of maleimides with different physico-chemical properties can be used, (6) the structure can be investigated under native conditions and does not require protein reconstitution into artificial bilayers.
...
PMID:A method for determining transmembrane protein structure. 914 63

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.
...
PMID:Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament. 919 94

The ability of xanthene dyes to mediate photoinduced reduction of nitrogenase was tested. In addition to eosin, which was studied in the preceding work (Biochemistry (Moscow), 1996, 61, 2165-2172), 4', 5'-dibromofluorescein (DBF), cyanosine, and erythrosin are effective photodonors of an electron in the presence of NADH. Fluorescein, rhodamine B, rhodamine 6G, and porphyrins are unable to mediate photoinduced reduction of nitrogenase. The mechanism underlying different efficiency of xanthene dyes in this reaction was studied. At high concentrations, all xanthene dyes tested were shown to inhibit the intramolecular electron transfer in nitrogenase. The inhibiting concentration of DBF is 1.5.10-4 M, whereas for other dyes, the inhibiting concentrations are less than 1.5.10-4 M. Under otherwise identical conditions, the ATPase activity was inhibited by xanthene dyes to a lesser extent than the nitrogenase activity. DBF, the most effective photodonor, was also studied by differential kinetic pulse laser spectroscopy. Photoinduced reduction of nitrogenase, (Fe-proteinox.Mo-Fe-protein).MgATP or (Av2ox.Av1).MgATP, was studied within the time range from 0 to 100 msec. Two initial stages of the nitrogenase turnover were detected: photoinduced reduction of Av2 and electron transfer from Av2red to Av1. The kinetics of the photoinduced reduction of Av2.MgADP was studied in the presence of DBF (up to 1.3.10-4 M) both in solution and the complex with Av1. The apparent second-order rate constants of the photoinduced reduction of Av2.MgADP in solution and the complex with Av1 were determined as 9.7.107 +/- 106 and 1.2.108 +/- 1.2.107 M-1. sec-1, respectively. The rate constant of the second reaction in the presence of another donor (dithionite) is 2500 times less. In complexes with Av1, the photochemical donor system DBF--NADH reduces Av2 more effectively than in free state in solution. In the presence of the photochemical donor system, neither photoreduction of Av2 in complexes with Av1 nor electron transfer from Av2red to Av1 are the rate-limiting stages of nitrogenase turnover.
...
PMID:Xanthene dyes as photochemical donors for the nitrogenase reaction. 976 91

ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.
...
PMID:Affinity labeling of two nucleotide sites on Na,K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[alpha-32P]diphosphate (TNP-8N3-[alpha-32P]ADP) as a photoactivatable probe. Label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate. 983 64

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.
...
PMID:Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic. 992 Jul 61

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.
...
PMID:Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase. 1008 Nov 49

The currently accepted topographical model for the organization of the alpha-subunit of the Na+, K+-ATPase in the membrane considers that the protein has ten transmembrane segments and six cytoplasmic loops. Evidence of interaction between the cytoplasmic regions may contribute to a better understanding of the structure/function relationship of this protein. In this study, the first four cytoplasmic segments (C1, C2, C3 and C4) of the rat alpha1 subunit were expressed in Escherichia Coli. The large cytoplasmic loop between transmembrane segments four and five (C3) retained its native structure as demonstrated by the ability of ATP to protect against chemical modification by Fluorescein 5-isothiocyanate (FITC). Interaction studies were conducted by an overlay assay (Far Western blots) and surface plasmon resonance technology. We observed that C3 interacts with the N-terminal segment of the Na+, K+-ATPase, C1; and that both C1 and C3 interact with the cytoplasmic segments C2 and C4.
...
PMID:Cytoplasmic segment interactions in the alpha1-subunit of the rat Na+, K+-atpase. 1147 30

The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.
...
PMID:The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site. 1537 38

The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10His-tagged fusion proteins. Both proteins exhibit a mainly alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na(+),K(+)-ATPase and Ca(2+)-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.
...
PMID:Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli. 1537 67


<< Previous 1 2 3 4 Next >>

---