Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+

Curcumin, an important inhibitor of carcinogenesis, is an inhibitor of the ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR). Inhibition by curcumin is structurally specific, requiring the presence of a pair of -OH groups at the 4-position of the rings. Inhibition is not competitive with ATP. Unexpectedly, addition of curcumin to SR vesicles leads to an increase in the rate of accumulation of Ca(2+), unlike other inhibitors of the Ca(2+)-ATPase that result in a reduced rate of accumulation. An increase in the rate of accumulation of Ca(2+) is seen in the presence of phosphate ion, which lowers the concentration of free Ca(2+) within the lumen of the SR, showing that the effect is not passive leak across the SR membrane. Rather, simulations suggest that the effect is to reduce the rate of slippage on the ATPase, a process in which a Ca(2+)-bound, phosphorylated intermediate releases its bound Ca(2+) on the cytoplasmic rather than on the lumenal side of the membrane. The structural specificity of the effects of curcumin on ATPase activity and on Ca(2+) accumulation is the same, and the apparent dissociation constants for the two effects are similar, suggesting that the two effects of curcumin could follow from binding to a single site on the ATPase.     

Change in the passive influx of Ca2+ into sarcoplasmic reticulum vesicles as a result of chemical modification of surface amino groups

Treatment of sarcoplasmic reticulum (SR) vesicles with trinitrobenzene (TNBS) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) stimulates the initial rate of passive influx of Ca2+ into SR vesicles, but does not affect either the binding or the maximal passive loading of SR vesicles with Ca2+. The changes in the kinetics of KCl-stimulated passive influx of Ca2+ depend on the reagent used. It is supposed that stimulation of passive influx of Ca2+ into SR vesicles and the changes in the reaction kinetics may be caused by modification of the Ca2+ channel gating behaviour as a result of binding of surface amino groups.     

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.     

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.     

A calcium pump made visible

The first high-resolution structure of a P-type ATPase, that of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, was published in 2000. This structure has provided many clues to how the Ca(2+)-ATPase might work, but no complete answers. The Ca(2+)-ATPase structure reveals no clear pathway from the cytoplasmic side of the membrane to the pair of high-affinity binding sites for Ca(2+) located in the transmembrane region of the ATPase and no clear pathway from these sites to the lumenal side of the membrane. The ATPase is therefore very unlike an ion channel in its construction. It is unclear from the crystal structure of the Ca(2+)-ATPase exactly how the protein sits within the lipid bilayer that surrounds it in the membrane. The Ca(2+)-ATPase is implicated in thermogenesis in some types of muscle; this could involve processes of slippage and leak modulated by interaction between the Ca(2+)-ATPase and sarcolipin.     

Charge changes in sarcoplasmic reticulum and Ca2+-ATPase induced by calcium binding and release: a study using lipophilic ions

Changes in the charge of sarcoplasmic reticulum (SR) vesicles are studied using lipophilic ions, which are adsorbed by the membrane phase. Upon addition of MgATP, phenyldicarbaundecaborane (PCB-) and tetraphenylboron (TPB-) are taken up by the SR vesicles, while tetraphenylphosphonium (TPP+) is released into the water phase. The PCB- uptake occurs as well under conditions when SR membrane is shunted by high Cl- concentration. MgATP induces minor additional binding of PCB- in the presence of oxalate and it is followed by release of the lipophilic anion from the vesicles. EGTA partly reverses the ATP effect, and calcium ionophore A23187 plus EGTA reverses it completely. Vesicles that were preliminarily loaded by Ca2+ demonstrated higher passive and lower ATP-dependent PCB- binding. Activation of isolated Ca2+-ATPase in the presence of 0.1 mM EGTA results in PCB- release into the medium and additional TPP+ binding to the enzyme. We suggest that the redistribution of the lipophilic ions between the water phase and SR membrane reflects charge changes in Ca2+-binding sites inside both SR vesicles and Ca2+-ATPase molecules in the course of Ca2+ translocation.     

Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.     

Characterisation of thapsigargin-releasable Ca(2+) from the Ca(2+)-ATPase of sarcoplasmic reticulum at limiting [Ca(2+)

The Ca(2+) binding sites of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using (45)Ca(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca(2+)-limited state, [Ca(2)](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca(2+)-ATPase, released a fraction of total Ca(2+) at [Ca(2+)](lim) that accumulated during active transport. Maximal Ca(2+) release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca(2+) resistant to TG. The amount of Ca(2+) released depended on the incubation time at [Ca(2+)](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0. 13 to 0.03 s(-1). The rapidly released early fraction declined with time and k=0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca(2+) followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 microM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca(2+) release mechanism. It is concluded that the Ca(2+) released by TG is from the occluded Ca(2+) fraction. The Ca(2+) occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca(2+) transport.     

Ca2+ -ATPase structure in the E1 and E2 conformations: mechanism,helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) in its Ca(2+)-bound [Nature 405 (2000) 647] and Ca(2+)-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca(2+)-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM alpha-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 A, rather than the normal 30 A. The experimentally determined TM alpha-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM alpha-helices in the Ca(2+)-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM alpha-helices, with changes in TM alpha-helical packing being important in the process of transfer of Ca(2+) ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM alpha-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca(2+)-ATPase.     

Hypothalamic factor inhibits the (Na,K)ATPase from the extracellular surface. Mechanism of inhibition

We have characterized the effect of a stable small molecule isolated from bovine hypothalamus (Haupert, G. T., and Sancho, J. M. (1979) Proc. Natl. Acad. Sci. 76, 4658-4660) on mammalian (Na,K)ATPase. This hypothalamus-derived inhibitory factor, HIF, has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity (Haupert, G. T., Carilli, C. T., and Cantley, L. C. (1984) Am. J. Physiol. 247, F919-F924). In this report it is shown that HIF inhibits the ouabain sensitive component of 86Rb+ uptake into human red blood cells. HIF also inhibited (Na,K)ATPase activity of unsealed red cell membranes but not that of sealed inside-out vesicles, indicating that HIF is impermeant to red cell membranes and inhibits the (Na,K)ATPase from the extracellular side. In unsealed human red cell membranes, concentrations of HIF which caused 70% inhibition of the (Na,K)ATPase did not inhibit ATP hydrolysis by plasma membrane (Ca2+)ATPase or (Mg2+)ATPase. However, at a similar concentration, HIF was shown to inhibit rabbit muscle sarcoplasmic reticulum (Ca2+)ATPase. HIF also inhibited p-nitrophenylphosphatase activity of unmodified or fluorescein-5'-iso-thiocyanate labeled dog kidney (Na,K)ATPase. As judged by fluorescein fluorescence of the modified enzyme, HIF stabilized the low fluorescent "E2" conformation of the enzyme similar to that stabilized by ouabain. However, unlike ouabain, HIF blocked covalent phosphorylation of dog kidney (Na,K)ATPase by inorganic phosphate. These studies show that HIF is an inhibitor of (Na,K)ATPase which acts from the extracellular side of the membrane by a mechanism similar to but not identical to that of cardiac glycosides.     

Influence of the 53 kDa glycoprotein on the cooperativity of the Ca(2+)-ATPase of the sarcoplasmic reticulum.

Previous results from this laboratory suggest that the 53 kDa glycoprotein (GP-53) of rabbit skeletal muscle sarcoplasmic reticulum membrane (SR) may influence coupling between Ca2+ transport and ATP hydrolysis by the Ca(2+)-ATPase. Here we report evidence that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase. The ATPase activity of the Ca(2+)-ATPase displays negative cooperative dependence (Hill coefficient n less than 1) on [MgATP] and has positive cooperative dependence (n greater than 1) on [Ca2+]free. We have determined the degree of cooperativity for native SR vesicles, SR preincubated with antiserum against GP-53 or preimmune serum, and SR partially extracted with KCl-cholate. Our results show that SR preincubated with preimmune serum or SR treated with cholate in 50 mM KCl (yielding membranes rich in GP-53) demonstrate a cooperative dependence of Ca(2+)-ATPase activity on both [ATP] and [Ca2+] similar to that of untreated SR. SR preincubated with anti-GP-53 antiserum (which causes an uncoupling of Ca2+ transport from ATP hydrolysis) or SR extracted with cholate in 1 M KCl (yielding membranes depleted of GP-53) displays decreased positive cooperative dependence on [Ca2+] and decreased negative cooperative dependence on [ATP]. The results are consistent with the interpretation that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase

Antibodies were produced against 5 peptides corresponding to segments of the (Ca2+-Mg2+)-ATPase of fast-twitch rabbit skeletal muscle sarcoplasmic reticulum (SR) including the N- and C-terminal regions. With the exception of antibodies directed against the peptide corresponding to residues 567-582 all antibodies bound strongly to the ATPase in intact SR vesicles, indicating that the epitopes were located on the cytoplasmic face of the SR. When the vesicles were disrupted, by solubilisation in SDS, binding of these antibodies was unchanged, further supporting the idea that these epitopes were located on the cytoplasmic face of SR. This is the first demonstration of the location of the N- and C-terminal regions of SR (Ca2+-Mg2+)-ATPase. These observations are discussed in the light of current structural models of the ATPase.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

Reconstitution experiments provide no evidence for a role for the 53-kDa glycoprotein in coupling Ca2+ transport to ATP hydrolysis by the (Ca(2+)-Mg2+)-ATPase in sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a 53 kDa glycoprotein of unknown function, as well as the (Ca(2+)-Mg2+)-ATPase. It has been suggested that the glycoprotein couples the hydrolysis of ATP by the ATPase to the transport of calcium. It has been shown that if SR vesicles are solubilized in cholate in media containing low K+ concentrations followed by reconstitution, then vesicles are formed containing the glycoprotein and with ATP hydrolysis coupled to Ca2+ accumulation, as shown by a large stimulation of ATPase activity by addition of A23187. In contrast, if SR vesicles are solubilized in media containing a high concentration of K+, then the vesicles that are produced following reconstitution lack the glycoprotein and show low stimulation by A23187 (Leonards, K.S. and Kutchai, H. (1985) Biochemistry 24, 4876-4884). We show that the effect of K+ on reconstitution does not follow from any changes in the amount of glycoprotein but rather from an effect of K+ on the detergent properties of cholate. In low K+ media, the cmc of cholate is high, cholate is a relatively poor detergent and incomplete solubilization results in 'reconstitution' of vesicles with the correct orientation of ATPase molecules. In high K+ media, the cmc of cholate is reduced and more complete solubilization of the SR leads to a true reconstitution with the formation of vesicles with a random orientation of ATPase molecules. The experiments provide no evidence for an effect of the glycoprotein on the (Ca(2+)-Mg2+)-ATPase.     

Calcium binding to the photosystem II subunit CP29

We have identified a Ca(2+)-binding site of the 29-kDa chlorophyll a/b-binding protein CP29, a light harvesting protein of photosystem II most likely involved in photoregulation. (45)Ca(2+) binding studies and dot blot analyses of CP29 demonstrate that CP29 is a Ca(2+)-binding protein. The primary sequence of CP29 does not exhibit an obvious Ca(2+)-binding site therefore we have used Yb(3+) replacement to analyze this site. Near-infrared Yb(3+) vibronic side band fluorescence spectroscopy (Roselli, C., Boussac, A., and Mattioli, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12897-12901) of Yb(3+)-reconstituted CP29 indicated a single population of Yb(3+)-binding sites rich in carboxylic acids, characteristic of Ca(2+)-binding sites. A structural model of CP29 presents two purported extra-membranar loops which are relatively rich in carboxylic acids, one on the stromae side and one on the lumenal side. The loop on the lumenal side is adjacent to glutamic acid 166 in helix C of CP29, which is known to be the binding site for dicyclohexylcarbodiimide (Pesaresi, P., Sandonà, D., Giuffra, E. , and Bassi, R. (1997) FEBS Lett. 402, 151-156). Dicyclohexylcarbodiimide binding prevented Ca(2+) binding, therefore we propose that the Ca(2+) in CP29 is bound in the domain including the lumenal loop between helices B and C.     

The plasma membrane H+-ATPase of Neurospora crassa. Properties of two reactive sulfhydryl groups

Previous work with N-ethylmaleimide (NEM) has defined two sites on the Neurospora plasma membrane H+-ATPase. Modification of one (the "fast" site) by NEM is rapid but does not affect ATPase activity, while modification of the other (the "slow" site) inactivates the enzyme and is protectable by MgATP or MgADP. In the present study, a wider array of sulfhydryl reagents have been used to examine the properties of both sites. The results show the following. (a) Both fast and slow sites react preferentially with hydrophobic compounds (N-pyrenemaleimide, dithiobisnitropyridine greater than N-naphthylmaleimide, dithiobisnitrobenzoate greater than N-phenylmaleimide greater than N-ethylmaleimide) and are virtually insensitive to hydrophilic sulfhydryl reagents such as iodoacetamide and iodoacetic acid. (b) The reaction rate of the slow site with NEM is approximately 2000-fold less rapid than that of the fast site. The slow site also has an unusually high pKa (greater than 9.5). (c) Whether or not cysteine modification leads to inactivation of the ATPase depends upon the site and the reagent. For example, when the fast site reacts with NEM, enzymatic activity is retained; when it reacts with N-pyrenemaleimide, activity is lost. Likewise, when the slow site is modified by any of the maleimides or by dithiobisnitropyridine or dithiobisnitrobenzoate, the ATPase is inactivated; when it is modified by methylmethanethiosulfonate, activity remains intact. Thus, neither cysteine can be considered to play an essential role in the reaction cycle of the ATPase, but the introduction of a sufficiently bulky substituent at either site can disrupt activity. (d) Upon reaction of methylmethanethiosulfonate at the slow site, the K1/2 for MgATP hydrolysis is reduced from 0.65 to 0.25 mM. This result strengthens the evidence for a conformational relationship between the slow site cysteine and the nucleotide binding site of the ATPase.     

Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites.

Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.     

Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle

The effect of gadolinium ions on the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptor (RyR1) was studied using heavy SR (HSR) vesicles and RyR1 isolated from rabbit fast twitch muscle. In the [(3)H]ryanodine binding assay, 5 microM Gd(3+) increased the K(d) of the [(3)H]ryanodine binding of the vesicles from 33.8 nM to 45.6 nM while B(max), referring to the binding capacity, was not affected significantly. In the presence of 18 nM[(3)H]ryanodine and 100 microM free Ca(2+), Gd(3+) inhibited the binding of the radiolabeled ryanodine with an apparent K(d) value of 14.7 microM and a Hill coefficient of 3.17. In (45)Ca(2+) experiments the time constant of (45)Ca(2+) efflux from HSR vesicles increased from 90.9 (+/- 11.1) ms to 187.7 (+/- 24.9) ms in the presence of 20 microM gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic (cis) (IC(50) = 5.65 +/- 0.33 microM, n(Hill) = 4.71) and the luminal (trans) side (IC(50) = 5.47 +/- 0.24 microM, n(Hill) = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 microM to 1 mM Ca(2+) concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd(3+) was applied at the trans side, EGTA was present at the cis side to prevent the binding of Gd(+3) to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd(3+) accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd(3+) exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd(3+) on both sides of the RyR1 with similar Hill coefficients and IC(50) values.     

Prolonged exercise induces structural changes in SR Ca(2+)-ATPase of rat muscle.

Sarcoplasmic reticulum (SR) isolated from the deep red portion of the gastrocnemius muscle of Sprague-Dawley rats after a single bout of prolonged exercise was shown to have depressed Ca(2+)-stimulated Mg(2+)-dependent ATPase activity over a temperature range of 15 to 42.5 degrees C when compared to SR obtained from control muscle. Inclusion of the calcium ionophore, A23187, failed to restore the depressed ATPase activity from SR of exercised muscle to control values, but it did normalize the stimulatory effect of temperature on ATPase activity. This depression was also manifested as an increased activation energy when the data were converted to an Arrhenius plot. SR vesicles from both groups showed no differences or discontinuities in plots of steady-state fluorescence anisotropy. When the binding characteristics of the fluorescent probe, fluorescein isothiocyanate (FITC), were analyzed, SR vesicles prepared from exercised muscle displayed a 40% reduction in binding capacity with no apparent change in Kd. These findings support the conclusion that a single bout of exercise induces a structural change in the Ca(2+)-ATPase protein of rat red gastrocnemius muscle that is not a direct result of gross lipid alterations or increased muscle temperature.