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1.
It has been shown previously that glutaraldehyde cross-links the Ca(2+)-ATPase of sarcoplasmic reticulum intramolecularly at the active site, involving residues participating in nucleotide binding and the conformational change that results in Ca2+ release to the vesicle lumen and formation of ADP-insensitive E2-P (Ross, D. C., Davidson, G. A., and McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621). This study shows that 10 nmol of [14C]glutaraldehyde/mg of protein attached irreversibly to the ATPase under conditions optimal for formation of the intramolecular cross-link. Half of this amount (i.e. 1 mol/mol ATPase) was inhibited by nucleotide binding. Thermolysin digestion of derivatized vesicles released two nucleotide-sensitive 14C-labeled species, which were isolated and identified as FSRDR*S AND FSRDR*S FA* FA*VEPS where the missing residues are Lys-492 and Arg-678. The majority of the 14C label was released in the sixth cycle of both Edman degradations, confirming the cross-link position. Lys-492 and Arg-678 are evidently close together in the active site, but their distance apart in the linear sequence suggests that they may arise from separate domains, which together constitute an ATP binding cleft. Residues in both regions, and Lys-492 in particular (McIntosh, D.B., Woolley, D.G., and Berman, M.C. (1992) J. Biol. Chem. 267, 5301-5309), have been derivatized by nucleotide-based affinity probes. Mutations of both of these residues in some of the bacterial P-type ATPases suggest that they do not play an essential catalytic role, and the inability of the cross-linked ATPase to form E2-P and to release Ca2+ to the lumen is probably because an essential tertiary structural movement at the active site is blocked.  相似文献   

2.
We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.  相似文献   

3.
The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.  相似文献   

4.
Phospholipase A2 (PLA2) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.  相似文献   

5.
Phospholipase A2 (PLA2) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.  相似文献   

6.
In most bacterial cell types studied, low intracellular free calcium is maintained by a variety of secondary exchangers which utilize transmembrane ion gradients. Prokaryotic calcium ATPases appear to be extremely uncommon, and none have been reported in Gram-negative organisms. We demonstrate ATP-dependent calcium uptake in everted membrane vesicles of Flavobacterium odoratum, a common Gram-negative soil and water bacterium. Calcium is transported with an apparent initial rate of 10 nmol/min mg of protein. It is inhibited by 20 microM orthovanadate, a specific P-type ATPase inhibitor, but significantly, it is unaffected by the addition of N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, valinomycin, or nigericin. Because the Ca(2+)-ATPase makes up a high proportion of the total ATPase activity it is easily detected by a soluble ATP hydrolysis assay, with an initial rate for calcium-dependent ATPase activity in vesicles of 25-40 nmol/min.mg at pH 7.8 and 25 degrees C. The calcium-dependent activity is preferentially solubilized by the detergent C12E8 and can be precipitated at 55-80% ammonium sulfate in a fraction free of other contaminating ATPase activities. This partially purified fraction is enriched 15-fold and demonstrates an apparent Km for calcium of 2 microM, and for ATP of 130 microM. The IC50 for vanadate is 1.6 microM. These values are similar to those obtained for the eukaryotic sarcoplasmic reticulum calcium ATPase. The enzyme is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent, vanadate-inhibitable manner. The phosphorylated species migrates with an apparent molecular mass of 60 kDa by NaDodSO4-polyacrylamide gel electrophoresis, and the phosphoryl group is sensitive to alkaline conditions, a characteristic of the acylphosphate linkage found in ATPases. These data demonstrate that the majority of calcium transport in F. odoratum is facilitated by a P-type ATPase.  相似文献   

7.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.  相似文献   

8.
Covalent adduct--the product of intracomplex alkylation at N-3-position of dC-8-nucleoside residue of target octanucleotide pd[TGTTTGGC] was completely synthesized by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5'-phosphamido derivative of heptanucleotide pd[CCAAACA]. Its melting temperature was shown to be 70 degrees C. Tm did not depend on covalent adduct concentration and was by 40 degrees C higher than that for unmodified duplex pd[TGTTTGGC].pd[CCAAACA] at concentration of 0.5 x 10(-4) M. The spatial structure of the covalent adduct in aqueous solution was investigated by two-dimensional 3H-NMR spectroscopy. The assignment of oligonucleotide protons as well as protons of a modifying group was carried out using COSY, COSY-DQF and NOESY experiments. Conformational analysis of proton-proton coupling constants for H1', H2'a, H2'b and H3' protons showed the sugar residues to be in 2'-endo conformation. Analysis of NOE connectivities observed between the protons of the alkylating group and oligonucleotide protons yielded conclusion, regarding the 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido 5'-residue being localized in the region of the lacked nucleoside residue of the heptanucleotide chain about 5 A apart from the dC-1 residue and from cytosine base of the alkylated dC-8 residue.  相似文献   

9.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.  相似文献   

10.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.  相似文献   

11.
S Hua  G Inesi 《Biophysical journal》1997,73(4):2149-2155
Sarcoplasmic reticulum (SR) Ca2+ ATPase was derivatized with 4,4'-diisothiocyanatostilbene-2,2'-sulfonic acid (DIDS), and complete enzyme inactivation was produced with a molecular stoichiometry of one DIDS per ATPase. It was determined by peptide analysis and sequencing that Lys492 and Lys515 were the ATPase residues derivatized by DIDS. Lack of electrophoretic resolution of the two peptide fragments that result from a single tryptic cut at Arg505 demonstrated that the two derivatized residues were cross-linked. Cross-linking of Lys492 and Lys515 by DIDS interfered with ATPase utilization of both ATP and p-nitrophenyl phosphate substrates, whereas derivatization of only Lys515 with fluorescein isothiocyanate interfered with ATPase utilization of ATP but not of p-nitrophenyl phosphate. Cross-linking with DIDS implies a distance of approximately 13 A between Lys492 and Lys515, which corresponds to the length of ATP bound in an extended configuration. Therefore, within the groove of the nucleotide binding domain, the ATP substrate is positioned with the adenosine moiety near Lys515 and its terminal phosphate near Lys492.  相似文献   

12.
Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.  相似文献   

13.
Hua S  Inesi G  Nomura H  Toyoshima C 《Biochemistry》2002,41(38):11405-11410
Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.  相似文献   

14.
The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.  相似文献   

15.
The catalytic subunit of sodium and potassium ion transport adenosine triphosphatase was isolated by sodium dodecyl sulfate-polyacrylamide electrophoresis and was subjected to isoelectric focussing on 3.5% acrylamide in 2% Triton X-100, 9 M urea, and 2% Bio-Lyte 3/10 from Bio-Rad Laboratories. At 20 degrees C this resolved 2 equal and closely spaced bands centered at pH 5.5 about 0.04 pH unit apart. The distribution of the polypeptide between the 2 bands came to a temperature-dependent equilibrium during focussing. At 15 degrees C predominantly the acidic band and at 25 degrees C predominantly the alkaline band appeared. Perhaps association of the nonionic detergent with the polypeptide resulted in its partitioning into bands corresponding to different physical states. A change of phase in a polypeptide-detergent complex might have altered its charge. To test functional homogeneity of the subunit in the native enzyme, the active center for ATP binding was covalently labeled with fluorescein isothiocyanate, an acidic ligand. Isoelectric focussing of the derivatized subunit at 20 degrees C showed displacement of all of the alkaline band to the position of the acidic band, which was fluorescent. Isoelectric focussing at 25 degrees C showed displacement of almost half of the alkaline band to the position of the acidic band, and both bands were fluorescent. The results suggest that all of the subunit accepted the fluorescent label and that derivatization slightly raised the temperature at which the polypeptide equilibrated between the 2 states. A few experiments on the calcium-dependent ATPase of sarcoplasmic reticulum indicated that it responded similarly.  相似文献   

16.
The radiolabeling of the natural octapeptide CCK8, derivatized with a cysteine residue (Cys-Gly-CCK8), by using the metal fragment [99mTc(N)(PNP3)]2+ (PNP3 = N,N-bis(dimethoxypropylphosphinoethyl)methoxyethylamine) is reported. The [99mTc(N)(NS-Cys-Gly-CCK8)(PNP3)]+ complex was obtained according to two methods (one-step or two-step procedure) that gave the desired compound in high yield. The complex is stable in aqueous solution and in phosphate buffer. In vitro challenge experiments with an excess of cysteine and glutathione indicate that no transchelation reactions occur, confirming the high thermodynamic stability and kinetic inertness of this compound. Stability studies carried out in human and mouse serum, as well as in mouse liver homogenates, show that the radiolabeled compound remains intact for prolonged incubation at 37 degrees C. Binding properties give Kd (19.0 +/- 4.6 nmol/l) and Bmax (approximately 10(6) sites/cell) values in A431 cells overexpressing the CCK2-R. In vivo evaluation of the compound shows rapid and specific targeting to CCK2-R, a fourfold higher accumulation compared to nonreceptor expressing tumors.  相似文献   

17.
The Ca(2+)-ATPase crystals formed in detergent solubilized sarcoplasmic reticulum (SR) at 2 degrees C in a crystallization medium of 0.1 M KCl, 10 mM K-Mops (pH 6.0), 3 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 20% glycerol and 20 mM CaCl2 (J. Biol. Chem. 263, 5277 and 5287 (1988)) contain highly ordered sheets of ATPase molecules, that associate into large multilamellar stacks (greater than 100 layers). When the crystallization is performed in the same medium but in the presence of 40% glycerol at low temperature the stacking is reduced to 4-5 layers and the average diameter of the crystalline sheets is increased from less than 1 micron to 2-3 microns. Glycerol and low temperature presumably reduce stacking by interfering with the interactions between the hydrophilic headgroups of Ca(2+)-ATPase molecules in adjacent lamellae, while not affecting or promoting the ordering of ATPase molecules within the individual sheets. Electron diffraction patterns could be regularly obtained at 8 A and occasionally at 7 A resolution on crystals formed in 40% glycerol, either at 2 degrees C or at -70 degrees C. In the same media but in the absence of glycerol, polyethyleneglycol 1450, 3000 and 8000 (1-8%) induced the formation of ordered crystalline arrays containing 10-12 layers that were similar to those obtained in 40% glycerol. Replacement of 40% glycerol with 10-50% glucose or supplementation of the standard crystallization medium with polyethyleneglycol (PEG 3000 or 8000; 1, 2, 5 and 8%) had no beneficial effect on the order of crystalline arrays compared with media containing 40% glycerol.  相似文献   

18.
The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-ATPase.  相似文献   

19.
The fluorescent probe, 6-acryloyl-2-dimethylaminonaphtalene (acrylodan) was reacted with the oligomycin-sensitivity conferring protein (OSCP). Acrylodan bound covalently to the single cysteinyl residue of the protein. Acrylodan-OSCP was fully competent in conferring oligomycin sensitivity to the mitochondrial F0-F1 ATPase complex. The fluorescence emission peak of acrylodan-OSCP was blue-shifted compared to that of an acrylodan-mercaptoethanol adduct, which means that acrylodan experiences a hydrophobic environment in OSCP. Binding of acrylodan-OSCP to the isolated F1 was accompanied by a red shift of fluorescence. It was achieved in less than 1 s at 25 degrees C. The titration curve revealed one high affinity OSCP binding site per F1. Acrylodan-OSCP appears to be an interesting tool for studying the dynamics of structural changes within the mitochondrial ATPase complex.  相似文献   

20.
4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.  相似文献   

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