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)

The structural properties of cardiac isomyosins from several species were compared using native gel electrophoresis, analysis of proteolytic digests, analysis of monoclonal antibody reactivity to specific proteolytic fragments on electroblots and S1 nuclease mapping with cDNA probes. The structure of specific regions of the myosin molecule was analyzed by reacting monoclonal antibodies with chymotryptic peptides of myosin separated by two-dimensional electrophoresis. The pattern of fragments reactive with antibody CCM-52 (epitope in LMM) was identical in all types of V3 isomyosin examined, and different in each type of V1 isomyosin. Peptides reactive with RCM-79 (epitope in HMM) were different from those reactive with CCM-52 and were also significantly different in each type of myosin examined. Thus, HC-alpha is structurally similar in the LMM portion of the molecule in all animals examined, while in the HMM region there are significant structural differences. HC-alpha differs from HC-beta, with structural differences in both LMM and HMM. We have also shown that atrial myosin HC and ventricular HC-alpha in the rabbit are indistinguishable both by RIA and peptide mapping analysis. The same conclusion was derived after analysis of the myosin HC mRNA expressed in rabbit atria and ventricles. Using cDNA probes specific for the alpha and beta myosin HC mRNA, we could not distinguish between the atrial myosin mRNA and ventricular HC alpha (V1 isomyosin) mRNA by S1 nuclease mapping experiments. Classification of different cardiac myosins is largely based on their mobility on native gel electrophoresis, immunological cross-reactivity, and ATPase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Classification and characterization of cardiac isomyosins. 653

The effects of 2,3-butanedione monoxime (BDM) and 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-me thy l-3,6-dihydro-2H-1,3,4-thiadiazin-2-one (EMD 53998) on cardiac muscle were studied in skinned muscle fibres from the right ventricle of the porcine heart. BDM decreases the Ca2+ sensitivity (pCa50 for 50% activation) and it exerts a dose-dependent inhibitory effect on force in troponin I (TnI)-depleted (unregulated) cardiac skinned muscle fibres (IC50 approximately 20 mM) thereby mimicking the effect of the TnI inhibitory peptide (cTnI 137-148, corresponding to the cardiac TnI inhibitory region) and that of inorganic phosphate (Pi). This inhibitory action can be antagonized by the calcium-sensitizing cardiotonic thiadiazinone derivative EMD 53998 that increases the IC50 to about 30 mM. In skinned fibres, BDM (10 mM) also increased the ratio of ATPase activity to isometric force (tension cost), whereas EMD 53998 (20 mu M) decreased it. We propose that BDM antagonizes EMD 53998 because both compounds affect the Pi release step of the crossbridge cycle in an antagonistic manner.
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PMID:The Ca2+ sensitizer EMD 53998 antagonizes the effect of 2,3-butanedione monoxime on skinned cardiac muscle fibres. 890 80

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 "mu"M), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 "mu"M and 150 "mu"M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.
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PMID:Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle. 892 1

Because of safety issues, components of the beta-adrenergic signaling pathway cannot currently be viewed as attractive targets for human gene therapy. Rather, the balance of evidence supports strategies that will target gene products specifically and directly at diastolic regulation. Augmenting the activity of the SR Ca2+ ATPase by AAV-mediated delivery of the SERCA2a gene, directed by a cardiac-specific promoter with a tightly regulable on-off switch is perhaps the most attractive strategy. PLB and cTnI also are attractive targets but only if molecular techniques can be devised to modulate their activity specifically and conditionally. Such techniques may involve modifying the phosphorylation sites in vitro and replacing wild type proteins in the failing heart with the modified forms, again using regulated AAV vectors for gene delivery.
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PMID:Molecular aspects and gene therapy prospects for diastolic failure. 1098 93

Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including alpha-actin, alpha-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of alpha-actin and alpha-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using beta-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca(2+)-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca(2+) relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.
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PMID:Functional consequences of caspase activation in cardiac myocytes. 1197 44

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.
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PMID:Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart. 1535 79

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnI(R145G) on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI(WT) or hcTnI(R145G). Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI(R146G) mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mm BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca(2)(+). Maximum force development (F(max)) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI(R145G) in vitro. Ca(2)(+) sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k(ACT) is reduced at maximal [Ca(2)(+)] but not at low [Ca(2)(+)] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k(REL). We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.
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PMID:Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils. 1571 66

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39 degrees C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39 degrees C than 33 degrees C. High ALP activity and mineralized nodule formation were observed at 39 degrees C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.
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PMID:Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene. 1598 73

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.
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PMID:A mutation in the N-terminus of troponin I that is associated with hypertrophic cardiomyopathy affects the Ca(2+)-sensitivity, phosphorylation kinetics and proteolytic susceptibility of troponin. 1632 98

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.
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PMID:Ala scanning of the inhibitory region of cardiac troponin I. 1948 81


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