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1.
The effects of E2 levuglandins on the contractile activity of rat uterine horns were studied. LGE2, AnLGE2, Δ9-LGE2 and the synthetic epimer, 8-epi-Δ9-LGE2 all induced contractions in a dose-response fashion. AnLGE2 gave decreased response with increased bath concentrations. Paired comparision showed potent and selective inhibitory effects of AnLGE2 on the uterotonic activity of prostaglandins. An LGE2 inhibited the uterotonic activity of PGe2 at a 0.1:1 ratio, of PGD2 at 1:1 ratio, but did not inhibit the activity of PGF. Exposure of sponteneously contracting uteri to high concentrations of AnLGE2, or prolonged exposure to lower concentrations, suppressed contractions.  相似文献   

2.
The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (ks) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (ks,max=5.57). The ks values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of ks, therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, ks,max=11.3 at pH 4; carbonic anhydrase from bovine, ks,max=6.53 at pH 4). The difference in the height and/or the position of the peaks of the ks–pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.  相似文献   

3.
Levuglandin E2 (LGE2) is a γ-keto aldehyde produced by rearrangement of the prostaglandin endoperoxide PGH2 under the aqueous conditions of its biosynthesis. We show that exogenous LGE2 enters cells and efficiently inhibits the first synchronous cell division of fertilized sea urchin eggs. We attribute this inhibition to covalent modification of tubulin and thereby to inhibition of microtubule assembly.  相似文献   

4.
Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2α and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2α > TXB2 > 6-keto-PGF1α, the stable degradation product of PGI2=PGD2=PGE2=13,14-dihydro-15-keto-PGF2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 PGD2 > 13,14-dihydro-15-keto-PGE2 > PGF2α=TXB2=6-keto-PGF1α > 13,14-dihydro- 15-keto-PGF2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.  相似文献   

5.
The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k1) and autocatalytic growth (A + B → 2B, rate constant k2). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k1) and growth (k2) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG, ΔH, and ΔS are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔGnucleation = 23(3) kcal/mol; ΔGgrowth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k1 value at pH ~ 3, as well as minimal k1 near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k2 values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k1 vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k1, nucleation step of α-synuclein. The chemically well-defined nucleation (k1) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k2 × [A]0 (where k2 is the rate constant for autocatalytic growth and [A]0 is the initial protein concentration) is related to the empirical constant, kapp obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.  相似文献   

6.
Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and thromboxane B2 (TXB2) were determined. The levels of AA, PGF2α, PGE2, 6-keto-PGF1α and TXB2 in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF2α, 6-keto-PGF1α and TXB2 were all significantly correlated to AA.These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF2α, PGE2 prostacyclin and thromboxane A2 in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.  相似文献   

7.
Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca2+ channels by an unknown mechanism at an unknown site. The Ca2+ channel pore-forming subunit (CaVα1) is a candidate for the site of AA inhibition because T-type Ca2+ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory CaVβ subunits on the inhibition of CaV1.3b L-type (L-) current by AA. Whole cell Ba2+ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β1b, β3, or β4 58, 51, or 44%, respectively, but with β2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes CaV1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of CaVβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for CaV2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β2a interfere with inhibition by AA. Our novel findings that the CaVβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that CaVβ expression may regulate how AA modulates Ca2+-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.  相似文献   

8.
Metabolism of [3H] arachidonic acid ([3H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [3H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [3H] AA to PGE2, PGF, PGFM, 6-keto-PGF, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [3H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE2 (12.3 ± 7.5) and 6-keto-PGF (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF2α (9.0 ± 4.1) and 6-keto-PGF (15.9 ± 2.7) than PGE2 (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF (4.0 ± 0.4) than PGF (0.5 ± 0.08) or PGE2 (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [3H] AA to determine the effects of indomethacin on [3H] AA metabolism. In general, indomethacin (Id; 4 × 10−4 M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [3H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [3H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.  相似文献   

9.
The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF, PGE2, PGD2, 6-keto-PGF and TXB2, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC50 = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC50 = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.  相似文献   

10.
Characteristics of xanthan gum-based biodegradable superporous hydrogel   总被引:1,自引:0,他引:1  
A novel biopolymer-based superporous hydrogel (SPH) was synthesized through chemical crosslinking by graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) on to xanthan gum (XG) via redox initiator system of ammonium persulfate (APS) and N, N, N′, N′-tetramethylethylenediamine (TMED), in the presence of N, N′-methylenebisacrylamide (MBA) crosslinking agent, sodium bicarbonate foaming agent, a triblock copolymer of polyoxyethylene/polyoxypropylene/polyoxyethylene as a foam stabilizer. Characterization of SPH was done by FT-IR, TGA, SEM, HPC and GCMS. The effects of pH and salinity on the swelling aptitude of the SPH were investigated along with its degradability in Streptococcus bovis medium.  相似文献   

11.
We have investigated the effects of norepinephrine (NE) and acetylcholine (ACh) on prostaglandin (PGE2 and 6 keto-PGF) production by rabbit iris, measured by radioimmunoassay (RIA), and the type of phospholipase activated by NE in irides in which phosphatidylinositol (PI) was doubly prelabeled with [3H] myo-inositol and [1-14C] arachidonic acid (14C-AA), quantitated by radiometric and chromatographic methods. PGE2 output in 60 min (3.6 μg/g tissue) was 2.6 times greater than 6 keto-PGF. PG production is time-dependent and it is stimulated by NE and ACh in a dose-dependent manner. The Ne- and ACh-induced release of PGE2, measured by RIA, is mediated through α1-adrenergic and muscarinic cholinergic receptors, respectively, and it requeires Ca2+ for maximal stimulation. Studies on the mechanism of AA release PI in irides doubly prelabeled with 14C-AA and [3H] myo-inositol revelased the following: (a) Both Ne and ACh increased the breakdown of PI, and this was accompanied by a significant increase in the release of AA and consequently PGE2. The stimulatory effects of NE and ACh are mediated through α1-adrenergic and muscainic cholinergic receptors respectively. (b) The NE-induced formation of 3H-lyso PI and the NE-induced metabolism of 14C-1,2-diacyl-glycerol (DG) are time-dependent. Two pathways for AA release from PI are probably operaitve in the iris: (a) An indirect release by PI-specific phospholipase C which producers DG, followed by the actions of DG- and monoacylglycerol lipases on DG to release AA. (b) A direct release by phospholipase A2. Whether lyso PI is a product of the polypholipids such as prosphatidylcholine and phosphatidylethanolamine could also serve as a source for AA in PG synthesis. In conclusion, the data presented provide evidence that in the iris the neurotransmitter-stimulated release of PG and AA, from phosphoinositides, for PG synthesis is coupled to the activation of α1-adrenergic and muscarinic cholinergic receptors.  相似文献   

12.
The 13C chemical shifts of several 85% 13C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH going from 7 to 1 all the δC are shifted more or less upfield with the exception of the carbonyl group of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal.The percentage of cis and trans conformers of glycyl-l-proline and glycyl-l-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-l-proline and only from 77 to 80% for glycyl-l-prolylglycine.The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-l-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the α-protons of the glycine residue, by a lower pK1 and by a larger ΔδHα of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-l-proline molecule.The 13C-13C coupling constants were also studied as a function of pH. The results show that JCo-Cα of a C-terminal residue, varying from 5 to 6 Hz and reflecting the pK of the carboxylate group, is a linear function of δCo and δCα as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the Cα−Co bond, however, is practically the same in both cases.Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and δCo, δCα on the one hand and the JCo-Cα on the other were established.  相似文献   

13.
The metabolism of arachidonic acid (AA) by caruncular and allantochorionic tissues and its regulation was studied in normal cows (n=13) and those with retained fetal membranes (RFM; n=9). Tissues were taken via the vagina about 6 hours postpartum and incubated for 6 hours in minimum essential medium containing tritiated AA alone or in the presence of oxytocin, platelet activating factor (PAF), epidermal growth factor (EGF) or ionophore calcium (A23187). The metabolites of AA were separated by reverse phase-high pressure-liquid chromatography. Tissue concentrations of prostaglandin F (PGF) and prostaglandin E2 (PGE2) and plasma 13,14-dihydro-15-keto-PGF (PGFM) concentration were also measured by radioimmunoassay. For caruncular tissue, less thromboxane B2 (TXB2) and more 6-keto prostaglandin F (PGIM) was synthesized in tissue from the animals with RFM than in the controls. Oxytocin, PAF, EGF and A23187 increased only PGIM production in the control animals; A23187 also decreased TBX2 synthesis. For the allantochorion, more PGE2, leukotriene B4 (LTB4) and PGIM and less TXB2, PGF and hydroxyecosatetranoic acids (HETE) was synthesized in tissue from cows with RFM than from animals that delivered normally. All of the substances used in this study increased PGIM, PGF and LTB4 and decreased TXB2 production by the allantochorionic tissue in control animals. The metabolism of AA by the allantochorionic tissue seems quantitatively under hormonal control. The metabolism of AA at the level of both maternal and fetal components of the placenta in cows with RFM differed from that seen in animals that expelled the membranes normally.  相似文献   

14.
In the mercuri- and halo-cyclizations of PGF2α methyl ester and its 11,15-bis(α-ethoxyethyl)-ether (or other protected forms) the exo-PGI1 derivative predominates independent of reagent and degree of protective of the PGF2α sample used. Diastereomerically pure samples of exo- and endo-PGI1 and prostacyclin (PGI2) were prepared. PGI0 epimers were prepared: catalytic hydrogenation of PGI2 Me ester provides exclusively the endo isomer. PGI2 methyl ester was found to be stable to extensive chromatography on silica, and to storage for at least a year in anhydrous ethanol at −20°C. At pH 7.4 in 2:1 H2O:EtOH, the ester has a half-life in excess of 5 hr at 25°C. A reproducible small scale (0.4–3 mg) synthesis of prostacyclin uses a modification of Whittaker's iodocyclization followed by DBN treatment. This procedure, developed with 15-3H-PGF2α, proved widely applicable to PGF2α analogs and diastereomers. The following prostacyclins (in the Me ester and Na salt forms) bearing the 5-en-6-yl ether unit were prepared in this way: ent-PGI2, rac-PGI2, 15-epi-PGI2, ent-15-epi-PGI2, 11-epi-PGI2, 8,9,12-epi-PGI2, -PGI2, 13,14-dihydro-PGI2, and 13,14-dihydro-15-epi-PGI2. NMR comparisons for the methyl esters reveal that of the resonances (H-5,9,11,15) that appear at δ4.0±0.6 ppm, the most deshielded is H-9 so long as the 5,6-olefin is . The 8 ,9 -6,9-oxido- -5,6-ene unit is most readily characterized by its strong positive dichroic absorption at 210–230 nm. CD spectroscopy not only serves to confirm the presence of this unit in analogs, but also can be used for quantitative analysis of PGI2 solutions and for monitoring the rate of hydrolytic cleavage of these enol ethers.  相似文献   

15.
Light scattering measurements were performed on dilute solutions of α-crystallin mixed with different combinations of βH, βL and γ-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of α-crystallin is progressively enhanced by its interactions with γ(βH+γ)(βH+βL+γ) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.  相似文献   

16.
Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (E0) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic EO injected animals (0.5+1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from E0 injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of E0 to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1α (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1α, PGF2α and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with E0 formed less 6-
amounts of PGF2α or of TXB2 from AA, than E0 injected controls, whereas uteri from castrated diabetic animals injected with E0, formed a similar % of 6-keto-PGF1α, PGF2α and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of E0 is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulatory problems.  相似文献   

17.
Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The α subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the Gα family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only 10% of total Gα protein expressed is active; the rest accumulates in inclusion bodies. Although G has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold Gα from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of Gα subunits (human Giα(1), human Gsα(short), human G11α and human Gtα(cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G expressed from E. coli showed that although the refolded Gα subunits were soluble and retained partial α-helicity characteristic of the native, folded Gα subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G protein has a native-like secondary structure, but is predominately in a molten globular state.  相似文献   

18.
The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied . After incubation of the whole membraneous cochlea with [3H]-arachidonic acid (AA), syntheses of PGF, 6-keto PGF, PGE2, thromboxane (TX) B2 and PGD2 were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10−5 M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37°C for 0–15 min, PGD2, PGE2, PGF2α and 6-keto PGF1α were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD2, PGF2α and 6-keto PGF1α, 90–120 pg/cochlea; PGE2, 370 pg/cochlea), reached to the maximum level (PGD2, PGF2α and 6-keto PGF1α, 170–200 pg/cochlea; PGE2, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB2 was lower than the detection limit by RIA (<50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE2 was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD2, PGF2α and 6-keto PGF1α were about of those of PGE2. In the LW, the amounts of PGD2, PGE2, PGF2α and 6-keto PGF1α were about the same level (70–100 pg/LW).  相似文献   

19.
Prostaglandin F2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F1α, F2β and F1β also cause the same cardiovascular effects as F2α, there is a decrease in potency for all parameters measured, with PGF2α>PGF1α>PGF2β>PGF1β. When compared to the actions of PGF2α in producing an increase in pulmonary arterial pressure, PGs F1α, F2β and F1β were less potent by approximately 10, 100, and 1000 fold respectively.  相似文献   

20.
The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acidsoluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, α and β, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the α and β subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an α3 β3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets [1] were the tetrameric and dimeric states of the molecule respectively.  相似文献   

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