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)

A general hypothesis for the molecular mechanism of membrane transport based on current knowledge of protein structure and the nature of ligand-induced protein conformational changes has recently been proposed [Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231]. According to this hypothesis, the essential reaction undergone by all proteinaceous transport catalysts is a ligand-induced hinge-bending-type conformational change that results in the transposition of binding-site residues from access on one side of the membrane to access on the other side. Subsequent release and/or alteration of the ligand or ligands that induce the conformational change facilitates the converse conformational change, which returns the binding-site residues to their original position. With this simple cyclic ligand-dependent gating process as a central feature, biochemically orthodox mechanisms for virtually all known transporters are readily conceived. In this article, a chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases of mitochondria, bacteria, and chloroplasts, formulated within the guidelines of this general transport paradigm, is presented. At least three points of potential interest arise from this exercise. First, with the aid of the model, it is possible to visualize how energy transduction catalyzed by these enzymes might proceed, with no major events left unspecified. Second, explicit possibilities as to the molecular nature of electric field effects on the transport process are raised. And finally, it is shown that enzyme conformational changes, energy-dependent binding-affinity changes, and several other related phenomena as well, need not be taken as evidence of "action at a distance" or indirect energy coupling mechanisms, as is sometimes assumed, because such events are also integral features of the mechanism presented, even though all of the key reactions proposed for both ATP-driven proton translocation and proton translocation-driven ATP synthesis occur at the enzyme active site.
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PMID:A chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases. 287 73

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.
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PMID:A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide. 295 48

Mammalian ventricle contains two major isomyosins, V1 and V3, which differ in the primary structure of their heavy chains (HC alpha alpha and HC beta beta, respectively) and in their adenosine triphosphatase activity. The distribution of the HC alpha isomyosin in the left ventricle of the rabbit was followed as a function of age and transmural location. HC alpha was detected with a monoclonal antibody found to be specific for the hinge region of V1 myosin molecules when viewed in the electron microscope after low-angle rotary shadowing. Frozen sections were observed with indirect immunofluorescence developed to this anti-HC alpha hinge antibody. Serial sections were observed with the histochemical assay for calcium-activated myosin adenosine triphosphatase, using preincubation at various pH levels. Results show that all the ventricular myocytes in baby rabbits (2 weeks) are stained by the HC alpha-antibody from the epi- to endocardium. The isomyosin content of myocytes varies through the epi- to endocardium of the right ventricular wall of the adult (1-year-old) rabbit, with the HC alpha form predominating in the outer epicardial third of the wall and the lowest amount of HC alpha in the middle third of the wall. A mixture of stained and unstained myocytes is seen in the endo- and subendocardial regions. The spatial distribution of HC alpha in 4-month-old rabbits varies between that of the baby and adult. There is good agreement between myocyte classifications made by histochemical and antibody staining methods.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transmural distribution of isomyosin in rabbit ventricle during maturation examined by immunofluorescence and staining for calcium-activated adenosine triphosphatase. 315 89

As a striated muscle, myocardium is characterized by a succession of contractile units, the sarcomeres, whose shortening is a consequence of the sliding of filaments. The thin filament bears the troponin-tropomyosin regulatory system. The main protein of the thick filament, myosin, is a hinge for the sliding, a receptor for actin and a catalytic site for ATP hydrolysis. This ATPase activity resulted from the combination of several isoenzyme forms of this polymer. Identification of these isoenzymes needed special precautions due to the high insolubility of myosin. The ATPase correlated with the speed of shortening as, for example, shown during chronic cardiac overload. In this condition the heart adapted to change in work by both increasing its mass and reducing its speed of shortening. The former corresponded to a stimulation of protein synthesis which affected equally myosin and actin and which was very likely a consequence of enhanced mRNA synthesis. The latter corresponded to an isoenzyme change of myosin to the benefit of the low ATPase form. This correlated with the maximal speed of shortening estimated on papillary muscle.
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PMID:[Cellular and molecular aspects of contraction in normal and hypertrophied heart]. 618 24

Saturation transfer electron paramagnetic resonance spectroscopy was used to investigate the rotational motion of the head domains of native and desensitized scallop myosin and its proteolytic subfragments. Scallop myosin was spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, which reacted with a heavy chain residue in the subfragment 1 domain. As previously shown for rabbit skeletal muscle myosin (Thomas et al., 1975), the two head domains of native scallop myosin appear to have independent motion (rotational correlation time, pi, = 0.8 X 10(-7) s for subfragment 1; 1.4 X 10(-7) s for myosin). However, removal of a regulatory light chain, to effect desensitization of the actin-activated ATPase, was associated with an increase in pi for myosin to a value of 2.4 X 10(-6) s. The Ca2+ sensitivity and initial correlation time were restored on recombination of the regulatory light chain in the presence of Mg2+. Sedimentation velocity profiles in an analytical ultracentrifuge indicated that the desensitized myosin preparations were largely monomeric and therefore the change in pi appears to reflect an intramolecular event. Addition of EDTA to spin-labelled scallop heavy meromyosin caused an immediate 2.5 to 4-fold increase in pi and a partial desensitization of the ATPase activity. Comparable experiments with subfragment 1 yielded a barely detectable increase in pi (1.5-fold) in the first ten minutes. The restricted rotational motion observed in desensitized myosin and heavy meromyosin could arise by a conformational change in the subfragment 1-subfragment 2 hinge region or by an association of one head with its partner. The latter mechanism, involving the exposed light chain binding site, would also explain the preferential release of one regulatory light chain from scallop myosin, and might account for some other co-operative effects observed in this molecule (Bagshaw, 1980).
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PMID:Segmental flexibility and head-head interaction in scallop myosin. A study using saturation transfer electron paramagnetic resonance spectroscopy. 630 70

We have determined the nucleotide sequence of the dnaB gene and the primary structure of the dnaB protein of Escherichia coli (Arai, K., Yasuda, S., and Kornberg, A. (1981) J. Biol. Chem. 256, 5247-5252). The coding region for the dnaB protein is 1413 base pairs followed by double stop codons and preceded by a possible promoter sequence. The dnaB gene lacks a typical Shine-Dalgarno sequence. The primary structure deduced from the DNA sequence is consistent with the protein chemical data. The dnaB protein contains 470 amino acid residues and has a calculated molecular weight of 52,265. In the mature protein, the initiator methionine residue is removed in vivo leaving alanine as the NH2-terminal residue. Based on the amino acid sequence, we predict that the dnaB protein may be composed of two domains. A hydrophilic NH2-terminal region (residues 1-20) is followed by a compact domain and a possible hinge region (residues 21-172) consisting primarily of alpha-helix. The sites of facile tryptic cleavage are at the arginine residues at 14 and 171. The DNA-dependent ATPase domain (residues 172-470) is located at the COOH-terminal end of the protein.
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PMID:Nucleotide sequence of dnaB and the primary structure of the dnaB protein from Escherichia coli. 632 20

The locations of ATP-induced structural change in gizzard myosin were examined by analyzing the changes in the chymotryptic fragmentation pattern. From the time course of fragmentation, and by fractionation of the produced fragments and detection of the masked amino terminal fragment, the original positions of the six different fragments in the parent myosin molecule were assigned. A reconstituted model of myosin based on the above assignment showed the presence of three cleavable sites in the heavy chain of myosin. ATP accelerated the cleavage at site 1, 5 K daltons from the masked amino terminus, while it inhibited cleavage at site 2, 71 K daltons from the N terminus. These opposite effects of ATP on sites 1 and 2 were remarkable, but ATP had no significant effect on cleavage at site 3, 100 K daltons from the carboxyl terminus. These results indicate that two distant regions in the myosin head, 66 K daltons apart in the primary sequence, were exposed or buried with the progress of the ATPase reaction. In addition, prolonged chymotryptic digestion failed to produce any subfragment-1, irrespective of the presence or absence of divalent cations in the digestion medium, but produced a protease-resistant and relatively long (100 K daltons) light meromyosin. This suggests a distinctive feature of the neck and hinge regions in gizzard myosin.
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PMID:Two opposite effects of ATP on the chymotryptic cleavages in smooth muscle myosin head. Determination of cleavable points and their characterization. 699 74

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

We have shown for the first time that the myosin head (subfragment-1, S1), the energy-transducing component in the actomyosin motor system undergoes a distinct shape change during hydrolysis of ATP using x-ray solution scattering techniques. Among various analogs for intermediate states of the S1 ATPase cycle, the complexes with MgADP and vanadate (S1.ADP.Vi), MgADP and beryllium fluoride (S1.ADP.BeF3), or MgADP and aluminum fluoride (S1.ADP.AIF4) showed a shape change similar to that in the presence of MgATP, but the complexes with ATP gamma S (S1.ADP gamma S) and MgADP trapped by cross-linking with pPDM (S1.ADP-pPDM) seemed to have a shape similar to that of nucleotide-free S1. These results indicate that the shape of an S1**.ADP.Pi state is more rounded or bent than in other intermediate states of the S1 ATPase cycle. Such changes occur in light chain 2-deficient S1 and also in smooth muscle S1. However, MgADP-fluoride complexes with smooth muscle S1 (without phosphorylation of a regulatory light chain) seemed to have a structure similar to that of nucleotide-free S1. Analysis of x-ray scattering data indicated that a conformational change of S1 in the presence of MgATP might be caused by a hinge-like bending movement between the catalytic and regulatory domains. The global change of S1 is correlated with some specific changes of a nucleotide-binding moiety.
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PMID:Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by x-ray solution scattering. 778 93

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.
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PMID:Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene. 780 2


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