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)

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart. 826 93

A genetic map of nine loci defined by polymorphic DNA markers was created using a single cross of F344/N and LEW/N rats. The markers contained polymorphic simple sequence repeats identified in five genes, renin (Ren), cardiac troponin T (Tnnt3), synaptotagmin (Syt2), Na+,K(+)-ATPase catalytic subunit (Atp1a2), and the Asp-, Gly-, Glu-, and Leu-tRNA gene cluster (Trnegl), as well as four anonymous DNA segments. Analysis of the segregation of the alleles of these markers in F2 intercross progeny of F344/N and LEW/N rats indicated the following locus order and distances between pairs of loci: D13N1-5 cM-Ren-1 cM-Tntt3-0 cM-Syt2-12 cM-D13N2-25 cM-Atp1a2-0 cM-Trnegl-7 cM-D13N3-4 cM-D13N4. Three of the loci, Ren, Trnegl, and Atp1a2, have previously been assigned to rat chromosome 13. Except for Ren, none of the loci have previously been mapped by linkage analysis. The markers for these loci were characterized in a total of 13 inbred rat strains (F344/N, LEW/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N) and were found to be highly polymorphic, with two to eight alleles detected for each marker. These markers expand the genetic map of the rat and should be valuable tools for future genetic studies. An examination of human and mouse comparative map information for all loci assigned to rat chromosome 13 shows significant synteny conservation with the q arm of human chromosome 1 and the distal portion of mouse chromosome 1.
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PMID:Linkage map of nine loci defined by polymorphic DNA markers assigned to rat chromosome 13. 828 30

Familial hypertrophic cardiomyopathy (HCM) can be caused by dominant missense mutations in cardiac troponin T (TnT), alpha-tropomyosin, C-protein, or cardiac myosin heavy chain genes. The myosin mutations are known to impair function, but any functional consequences of the TnT mutations are unknown. This report describes the in vitro function of troponin containing an IIe91Asn mutation in rat cardiac TnT, corresponding to the HCM-causing Ile79Asn mutation in man. Mutant and wild-type TnT cDNAs were expressed in bacteria and the proteins purified and reconstituted with the other troponin subunits, the mutation had no effect on troponin's affinity for tropomyosin, troponin-induced binding of tropomyosin to actin, cooperative binding of myosin subfragment 1 to the thin filament, CA(2+)-sensitive regulation of thin filament-myosin subfragment 1 ATPase activity, or the CA2+ concentration dependence of this regulation. However, the mutation resulted in 50% faster thin filament movement over a surface coated with heavy meromyosin in in vitro motility assays. The increased sliding speed suggests an unexpected role for the amino terminal region of TnT in which this mutation occurs. The relationship between this faster motility and altered cardiac contraction in patients with HCM is discussed.
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PMID:Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy. 867 96

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta-adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta-adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta-adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta-adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta-adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.
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PMID:Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy. 948 39

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.
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PMID:Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes. 953 95

Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity.
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PMID:Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy. 1008 22

Mutations in the gene encoding human cardiac troponin T can cause familial hypertrophic cardiomyopathy, a disease that is characterized by ventricular hypertrophy and sudden, premature death. Troponin T is the tropomyosin-binding subunit of troponin required for thin filament regulation of contraction. One mutation, a change in the intron 15 splice donor site, results in two truncated forms of troponin T [Thierfelder et al. (1994) Cell 77, 701-712]. In one form, the mRNA skips exon 16 that encodes the C-terminal 14 amino acids; in the other, seven novel residues replace the exon 15- and 16-encoded C-terminal 28 amino acids. The two troponin T cDNAs were expressed in Escherichia coli for functional analysis. Both C-terminal deletion mutants formed a complex with cardiac troponin C and troponin I that exhibited the same concentration dependence as wild-type for regulation of the actomyosin MgATPase. However, both mutants showed severely reduced activation of the regulated actomyosin in the presence of Ca2+, though the inhibition in the absence of Ca2+ and the Ca(2+)-dependence of activation were not altered. The C-terminal deletions reduce the effectiveness of Ca(2+)-troponin to switch the thin filament from the "off" to the "on" state. Both mutant troponin Ts have reduced affinity for troponin I; the shorter mutant is at least 6-fold weaker than wild-type. The low level of activation of the ATPase would be consistent with reduced contractile performance, and the results suggest reduced troponin I affinity may be the molecular basis for the disease.
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PMID:Altered regulatory function of two familial hypertrophic cardiomyopathy troponin T mutants. 1052 4

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.
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PMID:Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T. 1073 93

Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at least 8 contractile protein genes, most commonly beta myosin heavy chain, myosin binding protein C, and cardiac troponin T. Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion. To investigate the functional properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in Escherichia coli, purified, and combined with human cardiac troponins I and C to reconstitute human cardiac troponin. Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments motile and the velocity of motile filaments at pCa 9. (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocity of actin-tropomyosin filaments than wild-type troponin (12.3%). These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca(2+). However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca(2+) indicated an opposite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%). These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease.
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PMID:Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein. 1085 Sep 66

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


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