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1.
Three HCM-causing tropomyosin (Tm) mutants (V95A, D175N, and E180G) were examined using the thin-filament extraction and reconstitution technique. The effects of Ca 2+, ATP, phosphate, and ADP concentrations on cross-bridge kinetics in myocardium reconstituted with each of these mutants were studied at 25°C, and compared to wild-type (WT) Tm at physiological ionic strength (200 mM). All three mutants showed significantly higher (2–3.5 fold) low Ca 2+ tension ( TLC) and stiffness than WT at pCa 8.0. High Ca 2+ tension ( THC) was significantly higher for E180G than that for WT, whereas THC of V95A and D175N was similar to WT; high Ca 2+ stiffness ( YHC) had the same trend. The Ca 2+ sensitivity of isometric force was significantly greater for V95A and E180G than for WT, whereas that of D175N remained the same as for WT; for all mutants, cooperativity was lower than for WT. Nine kinetic constants and the cross-bridge distribution were deduced using sinusoidal analysis. The number of force-generating cross bridges was similar among the D175N, E180G, and WT Tm forms, but it was significantly larger in the case of V95A than WT. We conclude that the increased number of actively cycling cross bridges at pCa 8 is the major cause of Tm mutation-related HCM pathogenesis, which may result in diastolic dysfunction. Decreased contractility ( Tact) in V95A and D175N may further contribute to the severity of myocyte hypertrophy and related prognosis of the disease. 相似文献
2.
The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca 2+ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca 2+ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca 2+ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca 2+ release, with no significant change in SR Ca 2+ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA1), indicating that the reduced Ca 2+ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca 2+ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant. 相似文献
3.
The application of fluid pressure (FP) in ventricular myocytes using pressurized fluid flow inhibits L-type Ca 2+ current (I Ca), with approximately 80% of this effect coming through the enhancement of Ca 2+ releases from the sarcoplasmic reticulum. In the present study, we explored the remaining mechanisms for the inhibition of I Ca by FP. Since FP significantly increases H + concentration and H + is known to inhibit I Ca, we examined whether pH regulation plays a role in the inhibitory effect by FP on I Ca. A flow of pressurized (∼16.3 dyne/cm 2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes for which the I Ca was monitored using whole-cell patch-clamp under HEPES-buffered conditions. Extracellular application of the alkalizing agent, NH 4Cl (20 mM), enhanced I Ca by ∼34% in the control conditions while increasing I Ca significantly less (by ∼21%) in FP-pretreated myocytes, suggesting an inhibition of the effect of NH 4Cl on I Ca possibly by FP-induced acidosis. Application of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid, 500 μM), which blocks exchange but not Cl −–OH − exchange, did not alter the inhibitory effect of FP on I Ca. Replacement of external Cl − with aspartate attenuated the inhibitory effect of FP on I Ca. In highly Ca 2+-buffered cells, where Ca 2+-dependent inhibition of I Ca was minimized, the external Cl − removal eliminated the inhibitory effect of FP on I Ca. These results suggest that the decrease of I Ca in the presence of FP is at least partly caused by intracellular acidosis via activation of Cl −–OH − exchange in rat ventricular myocytes. 相似文献
4.
Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca 2+ binding and accumulating activities as well as (Mg 2+ + Ca 2+)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca 2+ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca 2+ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca 2+ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca 2+ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca 2+ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca 2+ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca 2+ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca 2+ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca 2+ accumulation ( V (nmol Ca 2+/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca 2+ required for half-maximal velocities did not differ significantly with age ( K0.5 for Ca 2+ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca 2+ binding also indicated that the velocity of Ca 2+ binding but not the concentration of Ca 2+ required for half-maximal binding was altered due to aging. At identical Ca 2+ concentrations, the combined Ca 2+ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca 2+ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca 2+ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg 2+ + Ca 2+)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca 2+ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca 2+ transport. Further, the age-related increment in the Ca 2+ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca 2+ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca 2+ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart. 相似文献
5.
The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca 2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants ( Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 10 6 L mol –1 and (5.26 ± 0.71) × 10 6 L mol –1, respectively. In addition, in the presence of calcium (10 μmol L –1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 10 7 L mol –1. In addition, the binding constant of KChIP4a with Ca 2+ was (7.1 ± 1.5) × 10 7 L mol –1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca 2+, and the integrity of the molecular structure of KChIP4a was important for Ca 2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming. 相似文献
6.
Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca 2+, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca 2+. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP. 相似文献
7.
We investigated the concentration- and Ca 2+-dependent effects of CaM mutants, CaM 12 and CaM 34, in which Ca 2+-binding to its N- and C-lobes was eliminated, respectively, on the Ca V1.2 Ca 2+ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM 12 and CaM 34 (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca 2+], suggesting that both functional lobes of CaM were necessary for the Ca 2+-dependent shift. However, channel activity induced by the CaM mutants showed Ca 2+-dependent decrease, implying a Ca 2+ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca 2+-dependent regulations of Ca V1.2 Ca 2+ channels. 相似文献
8.
Androcam is a calmodulin-like protein that acts as a testis-specific light chain to myosin VI during spermatogenesis in D. melanogaster. Modest, localized chemical shift changes that accompany Ca 2+ binding to the androcam N-terminal lobe indicate that unlike calmodulin, androcam does not undergo a dramatic conformational change upon binding calcium. Here we report the 1H, 15N and 13C resonances of androcam in the high calcium (10 mM) state and show the extent of chemical shift changes for backbone resonances relative to the low calcium state. 相似文献
9.
Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca 2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca 2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca 2+ influx that may lead to dysregulated cell growth in ADPKD. 相似文献
10.
Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca 2+-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca 2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca 2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca 2+ binding in EF-hand 4 and models the Ca 2+-free/Mg 2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca 2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca 2+-free activator and Ca 2+-bound inhibitor states. This contrasts with large Ca 2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1. 相似文献
11.
Type 1 ryanodine receptors (RyR1s) release Ca 2+ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca 2+ release response in HEK293 cells and bound the RyR-specific ligand [ 3H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K + conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca 2+ release in HEK293 cells, low [ 3H]ryanodine binding levels, and channels that were not regulated by Ca 2+ and did not conduct Ca 2+ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes. 相似文献
12.
To demonstrate the interaction of calpastatin (CS) domain L (CS L) with Cav1.2 channel, we investigated the binding of CS L with various C-terminus-derived peptides at ≈ free, 100 nM, 10 μM, and 1 mM Ca 2+ by using the GST pull-down assay method. Besides binding with the IQ motif, CS L was also found to bind with the PreIQ motif. With increasing [Ca 2+], the affinity of the CS L–IQ interaction gradually decreased, and the affinity of the CS L–PreIQ binding gradually increased. The results suggest that CS L may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca 2+-dependent manners. 相似文献
13.
Summary The divalent metal ion binding site and binding constant of ribonuclease HI from Escherichia coli were investigated by observing chemical shift changes using 1H– 15N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by 15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide 15N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg 2+, Ca 2+, or Ba 2+. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (K D=2–4 mM for the current divalent ions). 相似文献
14.
The cardiac sarcolemmal Na +-Ca 2+ exchanger is modulated by intrinsic regulatory mechanisms. A
large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A.
Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson.
1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has
an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties.
The major alteration was in a regulatory mechanism known as Na +-dependent inactivation. This inactivation is
manifested as a partial decay in outward Na +-Ca 2+ exchange current after application of Na + to the intracellular
surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was
markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na + affinities. Regulation of the exchanger by nontransported, intracellular Ca 2+ was also modified by the XIP-region
mutations. Binding of Ca 2+ to the intracellular loop activates exchange activity and also decreases Na +-dependent
inactivation. XIP-region mutants were all still regulated by Ca 2+. However, the apparent affinity of the group 1 mutants for regulatory Ca 2+ was decreased. The responses of all mutant exchangers to Ca 2+ application or removal
were markedly accelerated. Na +-dependent inactivation and regulation by Ca 2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement
of the exchanger into and out of the Na +-induced inactivated state, but that the XIP region is also involved in regulation by Ca 2+. 相似文献
15.
Increase of resting Ca 2+ levels and amplitude of vasopressin-induced Ca 2+ transients were observed when cells in serum-free medium were exposed to 5 mM Ca 2+ for 2 h. Small effect on cell viability was also observed. A rapid cytotoxic effect was developed in the presence of 10 mM Ca 2+ and absence of serum. However, cells exposed to 10 mM Ca 2+ in the presence of serum were protected from damage for at least 2 days. Resting Ca 2+ levels and cytosolic Ca 2+ transients in serum-containing medium with 10 mM Ca 2+ displayed lower increases and a tendency to recover control values. When serum was absent, cells preincubated with 10 mM Ca 2+ were more sensitive to thapsigargin-induced damage than cells preincubated with lower Ca 2+. The sensitivity was similar when serum was present. Tolerance to high Ca 2+ in the presence of serum was linked to potentiation of the mitochondrial Ca 2+ entry to decrease the sarcoplasmic reticulum Ca 2+ overload. 相似文献
16.
A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k
cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K
i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance. 相似文献
17.
Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca 2+. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca 2+ than with it. Ca 2+-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation. 相似文献
18.
Ca 2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl 2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca 2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca 2+ uptake, a second phase of Ca 2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca 2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca 2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca 2+ influx of sarcoplasmic reticulum near steady state of Ca 2+ uptake was measured by pulse labeling with 45Ca 2+. The Ca 2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca 2+ uptake in normal medium, Ca 2+ influx was balanced by Ca 2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca 2+ exchange rate at the first plateau of Ca 2+ uptake was about half of that in normal medium. When the second phase of Ca 2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca 2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca 2+ uptake was also observed. These data suggest that the Ca 2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca 2+ efflux, which subsequently stimulates Ca 2+ exchange. 相似文献
19.
Ca 2+ transport by the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) is sensitive to monovalent cations. Possible K + binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca 2+ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca 2+ transport correlated in most cases with their direct effects on SERCA. Choline +, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca 2+ transport. Of the monovalent cations tested, only Cs + significantly affected the Hill coefficient of Ca 2+ transport ( nH). An increase in nH from ∼2 in K + to ∼3 in Cs + was seen in all of the forms of SERCA examined. The effects of Cs + on the maximum velocity of Ca 2+ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K + binding sites of the different forms of the protein. 相似文献
20.
Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca 2+ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca 2+) and high affinity (50 µM Ca 2+) Ca 2+ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca 2+ ATPase activities measured at 1.25 mM and 4 mM Ca 2+ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca 2+ binding and Ca 2+ ATPase activities, whereas the methylation-induced effect on the high affinity Ca 2+ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca 2+ binding and Ca 2+ ATPase. A further decrease in Ca 2+ binding and Ca 2+ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na +-K + ATPase and Mg 2+ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca 2+ ATPase and low affinity ATP-independent Ca 2+ binding. 相似文献
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