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1.
Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.
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Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase. 相似文献
2.
Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography. 总被引:2,自引:2,他引:0
J A Chupa J P McCauley Jr R M Strongin A B Smith rd J K Blasie L J Peticolas J C Bean 《Biophysical journal》1994,67(1):336-348
X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A. 相似文献
3.
Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction. 总被引:2,自引:2,他引:0
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A number of studies have indicated that Ca(2+)-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) to these sites.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Monolayers of a model anesthetic-binding membrane protein: formation, characterization, and halothane-binding affinity
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Churbanova IY Tronin A Strzalka J Gog T Kuzmenko I Johansson JS Blasie JK 《Biophysical journal》2006,90(9):3255-3266
hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The alpha-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide. 相似文献
5.
Hydration state of single cytochrome c monolayers on soft interfaces via neutron interferometry
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Kneller LR Edwards AM Nordgren CE Blasie JK Berk NF Krueger S Majkrzak CF 《Biophysical journal》2001,80(5):2248-2261
Yeast cytochrome c (YCC) can be covalently tethered to, and thereby vectorially oriented on, the soft surface of a mixed endgroup (e.g., -CH3/-SH = 6:1, or -OH/-SH = 6:1) organic self-assembled monolayer (SAM) chemisorbed on the surface of a silicon substrate utilizing a disulfide linkage between its unique surface cysteine residue and a thiol endgroup. Neutron reflectivities from such monolayers of YCC on Fe/Si or Fe/Au/Si multilayer substrates with H2O versus D2O hydrating the protein monolayer at 88% relative humidity for the nonpolar SAM (-CH3/-SH = 6:1 mixed endgroups) surface and 81% for the uncharged-polar SAM (-OH/-SH = 6:1mixed endgroups) surface were collected on the NG1 reflectometer at NIST. These data were analyzed using a new interferometric phasing method employing the neutron scattering contrast between the Si and Fe layers in a single reference multilayer structure and a constrained refinement approach utilizing the finite extent of the gradient of the profile structures for the systems. This provided the water distribution profiles for the two tethered protein monolayers consistent with their electron density profile determined previously via x-ray interferometry (Chupa et al., 1994). 相似文献
6.
Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport. 总被引:3,自引:2,他引:1
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J K Blasie L G Herbette D Pascolini V Skita D H Pierce A Scarpa 《Biophysical journal》1985,48(1):9-18
X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer. 相似文献
7.
Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase on a 22,000-dalton protein, Phosphorylation is associated with an increase in both the initial rate of Ca2+ uptake and the Ca(2+)-ATPase activity which is partially due to an increase in the affinity of the Ca(2+)-Mg(2+)-ATPase (E) of sarcoplasmic reticulum for calcium. In this study, the effect of cAMP-dependent protein kinase phosphorylation on the binding of calcium to the SR and on the dissociation of calcium from the SR was examined. The rate of dissociation of the E x Ca2 was measured directly and was not found to be significantly altered by cAMP-dependent protein kinase phosphorylation. Since the affinity of the enzyme for Ca2+ is equal to the ratio of the on and off rates of calcium, these results demonstrate that the observed change in affinity must be due to an increase in the rate of calcium binding to the Ca(2+)-Mg(2+)-ATPase of SR. In addition, an increase in the degree of positive cooperativity between the two calcium binding sites was associated with protein kinase phosphorylation. 相似文献
8.
The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca(2+)-ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca(2+)-ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca(2+)-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca(2+)-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca(2+)-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca(2+)-ATPase is not a significant regulatory feature of cardiac Ca(2+) homeostasis. 相似文献
9.
我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。 相似文献
10.
A G Benders T H van Kuppevelt A Oosterhof R A Wevers J H Veerkamp 《Biochimica et biophysica acta》1992,1112(1):89-98
Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation. 相似文献
11.
We characterized the interaction of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) with the sarcoplasmic reticulum (SR) Ca(2+)-ATPase from rabbit fast-twitch skeletal and canine cardiac muscles by examining the effect of this agent on the ATPase reaction. tBuBHQ at less than 10 microM inhibited ATP hydrolysis by both isoforms of Ca(2+)-ATPase by up to 80 and 90%, respectively. The half maximal inhibition of these enzymes was observed at about 1.5 microM tBuBHQ. Thus, this agent potently inhibits the fast-twitch skeletal and slow-twitch skeletal/cardiac isoforms of SR Ca(2+)-ATPase. tBuBHQ at 5-10 microM inhibited the rate of decomposition of the phosphoenzyme intermediate (EP), measured as a ratio between ATPase activity and the EP level in the steady state, by 35-40%. It also inhibited formation of EP by decreasing the rate of Ca2+ binding to the Ca(2+)-deficient, nonphosphorylated enzyme to about 1/8 of the control value. These results indicate that tBuBHQ has at least two sites of action in the reaction sequence for the SR Ca(2+)-ATPase. 相似文献
12.
Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane. 总被引:1,自引:0,他引:1
Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes. 相似文献
13.
The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase. 相似文献
14.
Zhou L Burnett AL Huang PL Becker LC Kuppusamy P Kass DA Kevin Donahue J Proud D Sham JS Dawson TM Xu KY 《Biochemical and biophysical research communications》2002,294(5):1030-1035
Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart. 相似文献
15.
A Xu T Netticadan D L Jones N Narayanan 《Biochemical and biophysical research communications》1999,264(1):241-246
Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function. 相似文献
16.
The molecular environment of Ca2+ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca2+ + Mg2+)-ATPase has been studied by pulsed-laser excited luminescence of Eu3+ used as a Ca2+ analogue. Interaction of Eu3+ with SR was characterized by investigating its effect on partial reactions of the Ca2+ transport cycle. In native SR vesicles, Eu3+ was found to inhibit Ca2+ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca2+ uptake in parallel fashion. The non-specific binding of Eu3+ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca2+ + Mg2+)-ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca2+ transport by Eu3+ is due to its binding to Ca2+ translocating sites. The 7F0----5D0 transition of Eu3+ bound to these sites was monitored. The non-Lorentzian nature of the excitation profile and a double-exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H2O/D2O mixture buffers further distinguished the sites. The number of water molecules in the first co-ordination sphere of Eu3+ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process. 相似文献
17.
Logan-Smith MJ Lockyer PJ East JM Lee AG 《The Journal of biological chemistry》2001,276(50):46905-46911
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. 相似文献
18.
19.
The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
E M Reis E Kurtenbach A R Ferreira P J Biselli C W Slayman S Verjovski-Almeida 《Biochimica et biophysica acta》1999,1461(1):83-95
Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase. 相似文献