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1.
The characteristics of 3H-labeled imipramine and 3H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37°C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant (Kd) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas Kd for paroxetine is almost constant, about 0.05 nM, between 0 and 19°C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37°C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.  相似文献   

2.
Analysis of bacteriophage T7 early RNAs and proteins on slab gels   总被引:395,自引:0,他引:395  
The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn &; Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.  相似文献   

3.
Abstract A method proposed for estimating the rate of bacterial mortality in aquatic environments consists in following the disappearance of radioactive tracer from the macromolecular fraction of 3H-thymidine labeled natural assemblages of bacteria. The data presented in this paper offer a further technical validation of this procedure. Application of the method to North and Mediterranean Sea, estuaries, rivers and lakes yields first order mortality constant in the range 0.008–0.06 h−1. Mortality due to grazing by protozoans retained by 2 μm filtration range from 20 to 90% of the mortality detected by the method.  相似文献   

4.
Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs 3H-imipramine and 3H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both 3H-imipramine and 3H-mianserin. 3H-mianserin binding was potently displaced by serotonin S2 antagonists and exhibited a profile similar to that of 3H-spiperone binding. In the presence of the serotonin S2 antagonist spiperone, antihistamines (H1) potently displaced 3H-mianserin binding. 3H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing 3H-imipramine binding was not similar to their order in displacing 3H-spiperone or 3H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of 3H-imipramine but did not alter binding of 3H-mianserin. Binding of 3H-imipramine but not 3H-mianserin was sodium dependent. These results show that 3H-imipramine and 3H-mianserin bind to different receptors. 3H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. 3H-Mianserin binds to postsynaptic receptors, possibly both serotonin S2 and histamine H1 receptors, the binding of which is sodium independent.  相似文献   

5.
An evaluation of a number of commercial reactive textile dyes with regard to suitability for staining of proteins prior to polyacrylamide gel electrophoresis showed that a blue dye containing a difluorochloropyrimidyl group gave excellent quantitation. The sensitivity was as good as with other dyes suggested for the same purpose. Reaction conditions have been optimized to reach completion in 3 hr at 40°C. A simple device for slicing gels is described. The main errors responsible for experimental scatter are briefly discussed.  相似文献   

6.
The binding characteristics of [3H]-imipramine in slide mounted tissue sections of rat forebrain have been studied to ascertain the optimal binding conditions for labeling the sites prior to autoradiographic localization. The conditions for the experiments and the kinetics of the imipramine binding correspond reasonably well with those used in membrane preparations to initially define the imipramine binding site. Subsequent labeling of sections, using these parameters, allowed the autoradiographic localization of high concentrations of imipramine binding sites in such areas as the cerebral cortex, striatum, and several limbic and visual system structures. In addition, there was a marked overlap between regions demonstrating imipramine binding and areas known to be innervated by serotonergic neurons. This study outlines the potential sites of action of imipramine in the brain and defines areas for future investigations which attempt to localize brain regions involved in the etiology of depression and areas involved in the side effects of antidepressant drug therapy.  相似文献   

7.
In a previous study where rat skin collagen was labeled with 18O in the hydroxyl group of the collagen hydroxyproline we noticed that the decay rate of this label was much faster than had been observed when the skin collagen hydroxyproline was labeled with 3H in the prolyl ring. In this study a rat was labeled concurrently with [18O2] and [3H] proline and the rate of decline of both labels was determined in rat skin collagen hydroxyproline. After correction for growth dilution of the skin collagen the [18O] hydroxyproline was found to have a half-life of 27 days while the [3H] hydroxyproline had a half-life of 53 days. The decay rate of the [18O] hydroxyproline represents the true turnover rate of collagen since there is no possibility of recycling this label. Hence, the difference between this and the [3H] hydroxyproline decay rate is due to recycling of l-[3H] proline into new collagen. The efficiency of recycling of proline from catabolized collagen into new collagen was about 93%.  相似文献   

8.
Radioactive S-alkyl glycerol ethers have been synthesized with 35S, 14C and 3H labels as well as 3H/35S double labels.The synthesized compounds were converted to various derivatives which can serve to characterize the S-alkyl glycerol ethers. These included the isopropChemical analysis, IR, NMR, zonal TLC profile scans and GLC showed all the products to be > 99% pure.The GLC behaviour of the aldehyde and acetate derivatives of both S-alkyl glycerol ethers and O-alkyl glycerol ethers on EGSS-X was compared.  相似文献   

9.
A. Bobik 《Life sciences》1982,30(3):219-228
Binding of the alpha adrenergic antagonists (3H) prazosin and (3H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (3H) prazosin and (3H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order epinephrine > norepinephrinea?isoproterenol, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (3H) prazosin and (3H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (3H) yohimbine specific binding. The affinity of prazosin for the (3H) yohimbine binding site is approximately 2500 times less than for the (3H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (3H) yohimbine than (3H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (3H) yohimbine binding sites in different arteries vary about two fold whilst for (3H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha1 and alpha2 adrenoceptors.  相似文献   

10.
11.
With the use of 14C-arginine labelled basic proteins isolated from cytoplasma of Ehrlich ascites tumor cells it has been shown that these proteins are occluded by fibrin clots under the influence of thrombin. The occlusion of these proteins depends on their concentration. The experiments indicated that 2.5 μg of these proteins were occluded per one mg of fibrinogen in the presence of thrombin. The basic proteins occluded by fibrin make the clots resistant to the fibrinolityc action of plasmin. The clinical role of arginine-rich basic proteins appearing in circulation in malignancy have been discussed briefly.  相似文献   

12.
13.
Silver staining methods for protein detection in polyacrylamide gels have a quenching effect on autoradiography and fluorography. This effect was quantitated for proteins in two-dimensional gels by microdensitometry using a computer equipped with an image processor and by scintillation counting of proteins solubilized from the gels. The original histologically derived silver stain had a quenching effect that was severe and irreversible for 3H detection and moderate for 14C detection. A silver stain based on photochemical methods had minimal quenching of 14C detection and less of a quenching effect than the histological stain for 3H detection. The 3H quenching effect was partially reversible for the photochemical stain.  相似文献   

14.
Reaction of gibberellin A3 (GA3) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [3H]GA3 (1), [3H]GA1 (2) and [3H]tetrahydro GA3 (4). Another substance was isolated and tentatively identified as [3H]16,17-dihydro GA3 (3). GLC was used to determine the specific activities of 1 and 2. [3H]GA3 likely arises from palladium catalysed nonspecific exchange of GA3 alkane hydrogen atoms with tritium. [3H]GA1 is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA3.  相似文献   

15.
Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by 35S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with 3H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.  相似文献   

16.
The effect of ammonium (0.3 to 3.6 mM) was studied on the overflow of 3H-norepinephrine in the vas deferens and the brain of the rat. Ammonium enhanced the overflow of 3H-norepinephrine in both organs and this response was greatly facilitated by alkaline pH (7.8) and was blocked by pH 7.0. Calcium was not needed for ammonium-induced overflow. In fact, the overflow was exaggerated by the omission of calcium. Enhanced overflow of sympathetic neurotransmitter by ammonium, together with its modification by changes in pH, may be one of the factors responsible for the toxicities of hyperammonemia.  相似文献   

17.
We have built an electronic imaging system based on a modified charge-coupled-device television camera that directly quantitates the distribution of fluorescence from electrophoretic gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike the photographic system that it replaces, the response of the camera is directly proportional to the intensity of incident fluorescence, and image data are digitized and stored in computer memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand breaks in DNA.  相似文献   

18.
19.
Endothelial cells from injured frog corneas undergo increased 3H-uridine and 3H-actinomycin D (3H-AMD) incorporation as judged by autoradiography. The increase in 3H-AMD binding occurs when living endothelium is labeled in vitro or when fixed preparations are exposed to the drug. The changes in 3H-AMD incorporation detected by the two methods are comparable (55 and 62 % for living and pre-fixed tissue respectively). However, when fixed endothelium is also de-histonized with 2 N HCl, differential binding of 3H-AMD is eliminated. This result suggests that the enhanced incorporation of 3H-AMD into nuclei is at least partly due to a modification in the association of chromosomal proteins with DNA and not entirely to cell permeability changes that may accompany wound repair. This contrasts with observations of cells that are killed outright by the injury. Such cells bind very large amounts of 3H-AMD compared with their living neighbors. Here the difference in incorporation is eliminated by prefixation. Thus, in the dead cells increased binding may be due to a reduction of cell surface permeability barriers which accompanies cell morbidity.  相似文献   

20.
A procedure is described to quantitate inorganic phosphate in the form of the tris(trimethylsilyl) (TMS) phosphate by gas-liquid chromatography-mass spectrometry (glems) that increases the previously reported detection limit from the microgram to the nanogram range (i.e., 3 ng). The sensitivity for detecting TMS-phosphate by glc-ms analysis was shown to be limited by an increasing fractional loss with decreasing concentrations of TMS-phosphate analyzed due to its adsorption on different types of glc column supports. The method developed employs [18O4]phosphoric acid which serves as both an internal standard to permit quantitation and as a carrier to minimize sample adsorption on the glc column support.  相似文献   

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