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1.
(i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 mm, whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly. 相似文献
2.
A method is described for quantitative assay of cyanide which is based on measuring the rate of rise in absorbancy at 325 nm when cyanide forms an addition compound with NAD+ at pH 10. It was found that the rate of rise in absorbancy is proportional with the concentration of cyanide within the range of 0.05–4.00 mm. By applying this method it could be shown that in biological systems in which the interface between the aqueous phase and the atmosphere is large the effective concentration of cyanide, added to inhibit the respiration, decreases rather rapidly during incubation. 相似文献
3.
David Hartman Rudolf Werner 《Biochemical and biophysical research communications》1977,77(4):1445-1451
During the incorporation of α-[32P]-dATP into HeLa cell nuclei some of the [32P]-label enters ribonucleic acid presumably because of intracellular nucleotide turnover. This result makes the conclusion about the existence of an RNA-DNA covalent linkage, based on the transfer of [32P]-label from deoxyribonucleotides to ribonucleotides after alkaline hydrolysis of the nuclei acid, untenable. 相似文献
4.
A modification of the adenylyl cyclase assay method of Y. Salomon, C. Londos, and M. Rodbell (1974, Anal. Biochem. 48, 541–548) is described. It makes the method applicable to the determination of guanylyl cyclase in subcellular tissue fractions. The modification consists of performing the Dowex 50 chromatography at acid pH in a column that has bed volume that is three times as large. This modification results in low blanks and allows for reuse of both Dowex 50 and aluminum oxide columns. A modification of the method of Symons [1974, Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29, pp. 105–115, Academic Press, New York] for synthesis of [α-32P]nucleoside triphosphates is also presented. It allows all steps to be carried out within 5 h in a single reaction vessel. Synthesized NTPs are then purified by DEAE-Sephadex A-25 (HCO3?form) using a linear ammonium bicarbonate gradient. Its applicability to synthesis of [α-32P]GTP, [α-32P]dGTP, and [α-32P]ATP having specific activities of up to 75 Ci/mmol is shown. 相似文献
5.
Hexokinase (EC 2.7.1.1) will convert commercially available α-[32P]-labelled ATP into α-[32P]-labelled ADP. A simple, rapid isolation procedure for the α-[32P]-labelled ADP is described and this synthetic method can be used for the preparation of other α-[32P]-labelled nucleoside diphosphates. 相似文献
6.
8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca2+. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca2+ and Mg2+ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled. 相似文献
7.
《生物化学与生物物理学报:生物膜》2001,1510(1-2):378-400
Incubation of the isolated H+-ATPase from chloroplasts, CF0F1, with 2-azido-[α-32P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-32P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-32P]ATP was possible. CF0F1 with non-covalently bound 2-azido-[α-32P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF0F1. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F0-part and the F1-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385. 相似文献
8.
9.
Li Xu Da-wei Xiao Sheng Lou Jian-jun Zou Yu-bing Zhu Hong-wei Fan Guang-ji Wang 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2009,877(5-6):502-506
A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C18 column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H2O–H]?, m/z 331.1 for andrographolide and [M?H]?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers. 相似文献
10.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,885(2):195-205
1. The specific activity of the γ-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of 32P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-32P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-32P]ATP was 30–40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components. 相似文献
11.
Yohsuke Kikuchi Yusuke Naka Hidemitsu Osakabe Tetsuaki Okamoto Tomoko Masaike Hiroshi Ueno Shoichi Toyabe Eiro Muneyuki 《Biophysical journal》2013
Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α3β3γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH0 and ΔG0 values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α3β(Y341W)3γ subcomplex had similar Kd and ΔG0 values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy. 相似文献
12.
The improved methods for the preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2 were presented. The (α3+β2+)2 valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α2+β3+)2 valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α3+β2+)2 and (α2+β3+)2 valency hybrids. 相似文献
13.
Summary The 1.8-kb -globin gene fragments of DNAs from individuals heterozygous for nine different -thalassemia mutations involving 1, 2, 3, 4, or 25 basepair (bp) insertions or deletions were amplified by the polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on aqueous 8% polyacrylamide gel. In each heterozygote with either a 2 to 25 bp deletion, but not with a 1 bp insertion, two slower migrating bands representing heteroduplexes in addition to the 1.8-kb homoduplex band were seen. The electrophoretic positions of these slower migrating bands were characteristic of each mutation studied. By co-amplification with known normal DNA, it was also possible to distinguish DNAs from normal individuals and from individuals who are homozygous for the small insertion/deletion mutations. These studies demonstrate that the heteroduplex formation generated in PCR can be applied as a simple method in the diagnosis of insertion/deletion mutations involving 2 to 25 bp in -thalassemias as well as in other genetic disorders. 相似文献
14.
Hb Villaverde [β89 (F5) Ser → Thr], identified in a Spanish patient, is a new human hemoglobin variant, electrophoretically silent, responsible for a severe erythrocytosis. This abnormal hemoglobin displays a very high oxygen affinity and a markedly reduced cooperativity that is partly restored in the presence of IHP. Determination of the structural abnormality was achieved on a mixture of the normal and abnormal β-chains. After isolation of the abnormal tryptic peptide by RP-HPLC, its sequence was determined by mass spectrometry. The structural abnormality disturbs the intrasubunit interaction between helices F and H and, thus, may weaken the C-terminal bonds of the deoxy conformation and the heme contacts of several hydrophobic residues. Hb Villaverbe demonstrates that this intrasubunit contact between helices F and H is essential for the cohesion of the hemoglobin molecule. 相似文献
15.
José J. García Armando Gómez-Puyou Marietta Tuena de Gómez-Puyou 《Journal of bioenergetics and biomembranes》1997,29(1):61-70
Some of the characteristics of unisite hydrolysis of [32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site. 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,885(2):136-145
The phosphorylation of a protein of 80 kDa in permeable mouse lymphocytes is shown to be dependent both on exogenously added calcium and on concanavalin A. Lymphocyte plasma membranes are rendered permeable to exogenously added [γ-32P]ATP and other small molecules by treatment with 20 μg/ml α-lysophosphatidylcholine for 1 min on ice. Treated cells are permeable to Trypan blue dye and exhibit phosphatidylinositol turnover in response to concanavalin A stimulation. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, maximal phosphorylation of this protein occurs 5 min after addition of 20 μM calcium and 4 μg/ml concanavalin A. Exogenously added cyclic nucleotide cofactors do not enhance the phosphorylation of this 80 kDa protein, nor do inhibitors of calcium or calmodulin-dependent kinases suppress it, although in each case, other proteins are affected. In contrast, an inhibitor of the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), H-7, strongly suppresses the phosphorylation of the 80 kDa protein. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a known activator of protein kinase C, significantly increases the phosphorylation of the 80 kDa protein. Finally, this protein is phosphorylated at a serine residue. These results taken together suggest that it is a substrate for protein kinase C. The possibility that it may also be an element of the concanavalin A signal transduction mechanism is discussed. 相似文献
17.
18.
D.H. van den Eijnden D.H. Joziasse L. Dorland H. van Halbeek J.F.G. Vliegenthart K. Schmid 《Biochemical and biophysical research communications》1980,92(3):839-845
Partial sialylation of biantennary and triantennary glycopeptides of α1-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution 1H-NMR spectroscopic analysis of the isolated products enabled the assignment of the branch as the most preferred substrate site for sialic acid attachment. The branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal -acetyllactosamine units on the oligosaccharide chains of serum glycoproteins. 相似文献
19.
A model is described, which allows the determination of 95% confidence limits for the maintenance coefficient and the efficiency of oxidative phosphorylation for chosen values of the growth yield for ATP corrected for energy maintenance (Y
ATP
max
). As experimental data the specific rates of substrate consumption, product formation and oxygen uptake in chemostat cultures at various growth rates are used. 相似文献
20.
James M. Harrington 《Inorganica chimica acta》2005,358(15):4473-4480
The formation constant, log β4 = 62.3 for [Pd(CN)4]2− is reported at 25 °C in 0.1 M NaClO4. This value of log β4 was determined using a competition reaction, monitored using UV-Vis spectroscopy and 1H NMR. The competition reaction used was with the tetraamine ligand 2,3,2-tet(1,4,8,11-tetraazaundecane), for which log K1 = 47.8 at 25 °C in 0.1 M NaClO4 was determined by competition with thiocyanate, as described by earlier workers (Q.Y. Yan, G. Anderegg, Inorg. Chim. Acta 105 (1985) 121.). Also reported is a value of log β4 for the [Pd(SCN)4]2− ion of 27.2 in 0.1 M NaClO4, determined by competition with 2,2,2-tet. Measurement of log K1 for cyclam with Pd(II) was attempted using a competition reaction with cyanide, combined with the very high value of log β4 for [Pd(CN)4]2− measured here. It appeared that the equilibrium being followed was actually [Pd(cyclam)]2+ + 2CN− ? [Pd(cyclam)(CN)2], for which a constant of log K = 5.2 was obtained. 1H NMR and IR studies suggested that the complex [Pd(cyclam)(CN)2] was prone to oxidation to Pd(IV), followed by disproportionation to [Pd(cyclam)]2+ and, presumably, (CN)2. The very high value of log β4 for [Pd(CN)4]2− found here appears to be the highest formation constant known for any metal ion. 相似文献