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1.
Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment 总被引:3,自引:0,他引:3
E Goormaghtigh R Brasseur J M Ruysschaert 《Biochemical and biophysical research communications》1982,104(1):314-320
The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [3H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg2+ and EDTA were added, [3H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of so that [3H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of on antagonist binding but the effects of sodium and Gpp(NH)p on [3H]QNB binding were not additive. 相似文献
2.
Kelvin W. Gee Frederick J. Ehlert Henry I. Yamamura 《Biochemical and biophysical research communications》1982,106(4):1134-1140
The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [3H]FLU and [3H]propyl beta-carboline-3-carboxylate ([3H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [3H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10?4 M) on the competition curve was detected. In contrast, when assays were carried out at 0°C using [3H]FLU or a high concentration of [3H]PCC to achieve [3H]ligand receptor occupancy of both type I and type II receptors, GABA (10?4 M) caused a significant increase in the affinity of FLU as measured by and competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated. 相似文献
3.
Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane 总被引:17,自引:0,他引:17
A E David 《Journal of molecular biology》1973,76(1):135-148
Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with 14C-labeled amino acids, pulse-labeled with [3H]leucine and incubated for various times with an excess of non-radioactive leucine. The ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [3H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ratios of polypeptides in the plasma membrane did not reach the ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [3H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules. 相似文献
4.
Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated by ion-exchange chromatography. The artifactual results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the and []cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that is genuinely oxidized to , and that purification of substrate before its use is necessary. Production of and various []lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described. 相似文献
5.
Reynold Spector 《Archives of biochemistry and biophysics》1980,205(1):85-93
In vitro, the accumulation and release of [methyl-3H]thymidine ([]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of []thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated []thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the []thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm []thymidine in the medium, the choroid plexus accumulated []thymidine against a concentration gradient. However, the majority of the []thymidine within the choroid plexus was metabolized to []thymidine nucleotides at low extracellular []thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration. 相似文献
6.
David B. Glass Philip P. Dembure Jean H. Priest Louis J. Elsas 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,840(2):143-152
A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the α chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells. 相似文献
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9.
[14C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-14C]arginine or l-[guanidino-14C]canavanine. []Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-14C]canavanine. These metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [14C]hydroxyguanidine to [14C]guanidine, and by repeated recrystallization of isolated urinary []guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat. 相似文献
10.
N. Vaysse J. Laval C. Senarens J.P. Esteve A. Ribet 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4):378-383
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10?8 M and was half-maximal at . The increase at 1·10?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10?5 M dopamine was . Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The value for high affinity binding sites was and for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was noradrenaline, 2·10/t-7 M epinine, fluphenazine, haloperidol, cis-flupenthixol, trans-flupenthixol, apomorphine, sulpiride, naloxone and isoproterenol. 相似文献
11.
(1) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of 3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F1) was studied in the absence of photoirradiation, and compared to that of . The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by upon photoirradiation. (3) Photoirradiation of F1 by resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol . Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound was localized on the α- and β-subunits of F1. At low concentrations (less than 10 μM), bound was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F1. 相似文献
12.
The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-32P] ATP to [1-3H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-32P] ATP in biological samples. 相似文献
13.
Binding of (?)-[]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 108m?1 min?1 and 0.189 min?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (KD) of 5 to 7 nm. Bound (?)-[]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[]dihydroalprenolol were not affected by β1-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β1-responses in vitro, whereas β2-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β2-adrenergic receptor. 相似文献
14.
Blanche P. Alter Sabra C. Goff 《Biochemical and biophysical research communications》1980,94(3):843-848
Human embryonic, fetal, and adult globin chains () can be separated by electrophoresis on Triton Acid urea gels. K562, a human leukemia cell line, was induced with hemin, labelled with [3H]-leucine, and globin synthesis analyzed. All globins except β were produced. . Thus, hemin-induced K562 synthesized embryonic and fetal globin chains, and had globin synthetic imbalance, with “α-thalassemia.” 相似文献
15.
Nancy B. Schwartz Victoria Ostrowski Kenneth S. Brown Robert M. Pratt 《Biochemical and biophysical research communications》1978,82(1):173-178
Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [35S]-PAPS only or [35S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H235SO4. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. . (1976) Devel. Biol. , 82–94) may result from a defect in the synthesis of the sulfate donor PAPS. 相似文献
16.
An enzymatic method for [32P]phosphoenolpyruvate synthesis 总被引:7,自引:0,他引:7
A convenient method for the enzymatic synthesis of [32P]phosphoenolpyruvate from [γ-32P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-32P]ATP to [32P]phosphoenolpyruvate, which was subsequently separated from residual [γ-32P]ATP and by chromatography on AG-1-X8-bicarbonate resin. 相似文献
17.
Didier Pélaprat Jean-Marie Zajac Gilles Gacel Christiane Durieux Jean-Louis Morgat André Sasaki Bernard P. Roques 《Life sciences》1985,37(26):2483-2490
Preliminary results on the binding of [3H]Boc[Nle28,31]CCK27?33, designated [3H]Boc[diNle]CCK7, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK7 displaces [125I]CCK8 from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK8. The order of potencies to displace [3H]Boc[diNle]CCK7 from its binding sites was the same on mouse brain and rat pancreas: , the pancreas binding sites being more discriminative than the brain binding sites. Thus, [3H]Boc[diNle]CCK7 is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments. 相似文献
18.
A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na2SO4, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of , makes it possible to assay the synthesis of picomole amounts of [35S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [35S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[35S]phosphosulfate (PAPS) under the assay conditions adopted, so that the 35S remaining in solution after ethanol precipitation of is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [35S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme. 相似文献
19.
An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at followed by sedimentation of a microsomal fraction at . The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ( activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5–10 μM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell. 相似文献
20.
B S Jacobson C G Kannangara P K Stumpf 《Biochemical and biophysical research communications》1973,51(2):487-493
A disrupted spinach chloroplast preparation readily synthesized from [2-14C]acetate under anaerobic conditions. It can be shown by degradation data that [14C]oleate is not a precursor of [14C]linolenate and that 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [14C]linolenate. 相似文献