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)

To define the structural differences that are responsible for the functional diversity between orthologous sarcomeric myosins, we compared the rat and human beta/slow myosins. Functional comparison showed that rat beta/slow myosin has higher ATPase activity and moves actin filaments at higher speed in in vitro motility assay than human beta/slow myosin. Sequence analysis shows that the loop regions at the junctions of the 25 and 50 kDa domains (loop 1) and the 50 and 20 kDa domains (loop 2), which have been implicated in determining functional diversity of myosin heavy chains, are essentially identical in the two orthologs. There are only 14 non-conservative substitutions in the two myosin heavy chains, three of which are located in the secondary actin-binding loop and flanking regions and others correspond to residues so far not assigned a functional role, including two residues in the proximal S2 domain. Interestingly, in some of these positions the rat beta/slow myosin heavy chain has the same residues found in human cardiac alpha myosin, a fast-type myosin, and fast skeletal myosins. These observations indicate that functional and structural analysis of myosin orthologs with limited sequence diversity can provide useful clues to identify amino acid residues involved in modulating myosin function.
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PMID:Functional diversity between orthologous myosins with minimal sequence diversity. 1103 48

The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of this level during pre- and postmoult. Patterns of gene expression for SERCA and other sarcomeric proteins during the crustacean moulting cycle may be regulated by ecdysteroids and/or mechanical stimulation.
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PMID:Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase. 1104 80

We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules.
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PMID:Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules. 1117 81

Hypertrophic cardiomyopathy (HCM), a relatively common disease, is diagnosed clinically by unexplained cardiac hypertrophy and pathologically by myocyte hypertrophy, disarray, and interstitial fibrosis. HCM is the most common cause of sudden cardiac death (SCD) in the young and a major cause of morbidity and mortality in elderly. Hypertrophy and fibrosis are the major determinants of morbidity and SCD. More than 100 mutations in nine genes, all encoding sarcomeric proteins have been identified in patients with HCM, which had led to the notion that HCM is a disease of contractile sarcomeric proteins. The beta -myosin heavy chain (MyHC), cardiac troponin T (cTnT) and myosin binding protein-C (MyBP-C) are the most common genes accounting for approximately 2/3 of all HCM cases. Genotype-phenotype correlation studies suggest that mutations in the beta -MyHC gene are associated with more extensive hypertrophy and a higher risk of SCD as compared to mutations in genes coding for other sarcomeric proteins, such as MyBP-C and cTnT. The prognostic significance of mutations is related to their hypertrophic expressivity and penetrance, with the exception of those in the cTnT, which are associated with mild hypertrophic response and a high incidence of SCD. However, there is a significant variability and factors, such as modifier genes and probably the environmental factors affect the phenotypic expression of HCM. The molecular pathogenesis of HCM is not completely understood. In vitro and in vivo studies suggest that mutations impart a diverse array of functional defects including reduced ATPase activity of myosin, acto-myosin interaction, cross-bridging kinetics, myocyte contractility, and altered Ca2+ sensitivity. Hypertrophy and other clinical and pathological phenotypes are considered compensatory phenotypes secondary to functional defects. In summary, the molecular genetic basis of HCM has been identified, which affords the opportunity to delineate its pathogenesis. Understanding the pathogenesis of HCM could provide for genetic based diagnosis, risk stratification, treatment and prevention of cardiac phenotypes.
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PMID:The molecular genetic basis for hypertrophic cardiomyopathy. 1127 20

Ouabain-induced inhibition of early heart development indicated that Na/K-ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93-105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na(+)-Ca(++) exchanger (NCX) to increase intracellular [Ca(++)]. These previous findings suggested that Ca(++) signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca(++) regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca(++) ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca(++) modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo.
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PMID:Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development. 1145 86

Striated muscle tropomyosin (TM) interacts with actin and the troponin complex to regulate calcium-mediated muscle contraction. Previous work by our laboratory established that alpha- and beta-TM isoforms elicit physiological differences in sarcomeric performance. Heart myofilaments containing beta-TM exhibit an increased sensitivity to calcium that is associated with a decrease in the rate of relaxation and a prolonged time of relaxation. To address whether the carboxyl-terminal, troponin T binding domain of beta-TM is responsible for these physiological alterations, we exchanged the 27 terminal amino acids of alpha-TM (amino acids 258 -284) for the corresponding region in beta-TM. Hearts of transgenic mice that express this chimeric TM protein exhibit significant decreases in their rates of contraction and relaxation when assessed by ex vivo work-performing cardiac analyses. There are increases in the time to peak pressure and a dramatic increase in end diastolic pressure. In myofilaments, this chimeric protein induces depression of maximum tension and ATPase rate, together with a significant decrease in sensitivity to calcium. Our data are the first to demonstrate that the TM isoform-specific carboxyl terminus is a critical determinant of sarcomere performance and calcium sensitivity in both the whole heart and in isolated myofilaments.
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PMID:Functional importance of the carboxyl-terminal region of striated muscle tropomyosin. 1269 96

A depressed activity of myosin ATPase has been described in human failing myocardium. Since alterations in cross-bridge kinetics may affect both systolic and diastolic cardiac function, the present study simultaneously investigated Ca(2+)-dependent tension and actomyosin ATPase activity (MYO) in triton X-skinned fiber preparations of human non-failing (donor hearts, n=8) and failing (dilated cardiomyopathy, n=11) left ventricular myocardium at increasing sarcomeric length (1.9 and 2.1 microm, alpha-actinin staining). The MYO/tension ratio was analyzed as a parameter characterizing myofibrillar energetics. At a sarcomere length of 1.9 microm, the Ca(2+) sensitivity of tension was significantly increased in human failing compared to non-failing myocardium. In human non-failing myocardium, maximal Ca(2+)-activated tension [1.9 microm vs. 2.1 microm, 23.7 (1.9) vs. 28.3 (1.9) mN/mm(2)] and the Ca(2+) sensitivity of tension [EC(50)Ca(2+ )(pCa): 5.67 (0.06) vs. 7.07 (0.11)] were increased by increasing sarcomere length. This was accompanied by an enhancement in Ca(2+)-dependent MYO [+72 (11) vs. +101 (9) microM ADP/s] as well as an increase in the Ca(2+)-sensitivity of MYO [EC(50)Ca(2+ )(pCa): 5.84 (0.08) vs. 6.86 (0.08)]. In human failing myocardium, only Ca(2+) sensitivity of tension (but not of MYO) increased. Tension cost was increased in failing vs. non-failing tissue [1.9 microm: 4.18 (0.06) vs. 3.53 (0.06) (mN.s)/(mm(2). microM ADP); 2.1 microm: 4.28 (0.13) vs. 3.52 (0.05) (mN.s)/(mm(2). microM ADP)]. We concluded that, in human failing myocardium, the length-dependent force generation may be blunted due to an already increased Ca(2+) affinity of troponin C as well as an impairment of length-dependent cross-bridge recruitment.
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PMID:Reduced length-dependent cross-bridge recruitment in skinned fiber preparations of human failing myocardium. 1273 32

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 (2000) 24722]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca2+ handling observed in SERCA2(b/b), we wanted to compare SERCA2b distribution in SERCA2(b/b) with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2(b/b) mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca2+ handling and the development of hypertrophy in adult SERCA2(b/b) mice do not result from different SERCA2b targeting.
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PMID:Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart. 1457 4

The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca(2+) sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca(2+)-Mg(2+) binding site of HCRLC, which demonstrated no Ca(2+) binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca(2+) sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca(2+) binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC.
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PMID:Familial hypertrophic cardiomyopathy-linked alterations in Ca2+ binding of human cardiac myosin regulatory light chain affect cardiac muscle contraction. 1459 49

Familial hypertrophic cardiomyopathy (FHC) is associated with mutations in 11 genes encoding sarcomeric proteins. Most families present mutations in MYBPC3 and MYH7 encoding cardiac myosin-binding protein C and beta-myosin heavy chain. The consequences of MYH7 mutations have been extensively studied at the molecular level, but controversial results have been obtained with either reduced or augmented myosin motor function depending on the type or homogeneity of myosin studied. In the present study, we took advantage of the accessibility to an explanted heart to analyze for the first time the properties of human homozygous mutant myosin. The patient exhibited eccentric hypertrophy with severely impaired ejection fraction leading to heart transplantation, and carries a homozygous mutation in MYH7 (R403W) and a heterozygous variant in MYBPC3 (V896M). In situ analysis of the left ventricular tissue showed myocyte disarray and hypertrophy plus interstitial fibrosis. In vitro motility assays showed a small, but significant increase in sliding velocity of fluorescent-labeled actin filaments over human mutant cardiac myosin-coated surface compared to control (+18%; P<0.001). Mutant myosin exhibited a large increase in maximal actin-activated ATPase activity (+114%; P<0.05) and Km for actin (+87%; P<0.05) when compared to control. These data show disproportionate enhancement of mechanical and enzymatic properties of human mutant myosin. This suggests inefficient ATP utilization and reduced mechanical efficiency in the myocardial tissue of the patient, which could play an important role in the development of FHC phenotype.
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PMID:Human homozygous R403W mutant cardiac myosin presents disproportionate enhancement of mechanical and enzymatic properties. 1501 Feb 70


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