共查询到20条相似文献,搜索用时 31 毫秒
1.
We report ab initio calculations of the frequency-dependent electric dipole-magnetic dipole polarizabilities, beta(nu), at the sodium D line frequency and, thence, of the specific rotations, [alpha](D), of 2,7,8-trioxabicyclo[3.2.1]octane, 1, and its 1-methyl derivative, 2, using the Density Functional Theory (DFT) and Hartree-Fock/Self-Consistent Field (HF/SCF) methodologies. Gauge-invariant (including) atomic orbitals (GIAOs) are used to ensure origin-independent [alpha](D) values. Using large basis sets which include diffuse functions DFT [alpha](D) values are in good agreement with experimental values (175.8 degrees and 139.2 degrees for (1S,5R)-1 and -2, respectively); errors are in the range 25-35 degrees. HF/SCF [alpha](D) values, in contrast, are much less accurate; errors are in the range 75-95 degrees. The use of small basis sets which do not include diffuse functions substantially lowers the accuracy of predicted [alpha](D) values, as does the use of the static limit approximation: beta(nu) approximately beta(o). The use of magnetic-field-independent atomic orbitals, FIAOs, instead of GIAOs, leads to origin-dependent, and therefore nonphysical, [alpha](D) values. We also report DFT calculations of [alpha](D) for the 1-phenyl derivative of 1, 3. DFT calculations find two stable conformations, differing in the orientation of the phenyl group, of very similar energy, and separated by low barriers. Values of [alpha](D) predicted using two different algorithms for averaging over phenyl group orientations are in good agreement with experiment. In principle, the absolute configuration (AC) of a chiral molecule can be assigned by comparison of the optical rotation predicted ab initio to the experimental value. Our results demonstrate the critical importance of the choice of ab initio methodology in obtaining reliable optical rotations and, hence, ACs, and show that, at the present time, DFT constitutes the method of choice. 相似文献
2.
D R Kodali 《Journal of lipid research》1987,28(4):464-469
Optically active 1- or 3-acyl-sn-glycerols were synthesized from 2,3- or 1,2-isopropylidene-sn-glycerols, respectively. The 2,3- or 1,2-isopropylidene-sn-glycerols were condensed with appropriate long saturated or unsaturated fatty acids and the resulting acyl isopropylidene compounds were treated with dimethylboronbromide at - 50 degrees C to give the title compounds. The ketal cleavage of acyl isopropylidene-sn-glycerols by dimethylboronbromide to produce the long 1- or 3-acyl-sn-glycerols was effective and gave good yields (70-90%). The reaction conditions were mild and there was no acyl migration, as shown by optical rotation of the monoacyl-sn-glycerols. The synthesis of 2,3-isopropylidene-sn-glycerol was improved to give an overall yield of 40% from L-arabinose. L-Arabinose was first converted to its 1,1'-diethylmercapto derivative and then condensed with 2-methoxypropene to yield 1,1'-diethyl-mercapto-4,5-isopropylidene-L-arabinose. Oxidation of this compound with sodium periodate followed by reduction with sodium borohydride under alkaline conditions yielded 2,3-isopropylidene-sn-glycerol [alpha]22D = -14.90 degrees, neat (Lit. 8 [alpha]22D = -14.5 degrees, neat; 14 [alpha]25D = -10.8 degrees; methanol C, 16.9). The optical purity of isopropylidene-sn-glycerols was determined as benzoyl derivatives on a high performance liquid chromatographic column packed with a chiral stationary phase. 相似文献
3.
Resolution and adrenergic activities of the optical isomers of 4-[1-(1-naphthyl)ethyl]-1H-imidazole.
S S Hong K J Romstedt D R Feller F L Hsu C George T L Cupps R A Lyon D D Miller 《Chirality》1992,4(7):432-438
Recently we synthesized a naphthalene analog of medetomidine, 4-[1-(1-naphthyl)ethyl]-1H-imidazole hydrochloride (1), and found it to be highly potent in adrenergic systems. The separation of optical isomers of this naphthalene analog was achieved by using the isomers of tartaric acid. The optical purities of the isomers were determined by HPLC using a chiral column. Using X-ray analysis the (+)-isomer was determined to have the S absolute configuration. It has been reported that the (+)-isomer of medetomidine (2) is the most potent enantiomer on alpha 2-adrenergic receptors. There were both qualitative and quantitative differences in biological activities of the optical isomers of 1 in alpha 1- and alpha 2-adrenergic receptor systems of guinea pig ileum and human platelets. (+)-(S)-1, but not (-)-(R)-1 was a selective agonist of alpha 2-mediated responses in ileum whereas (-)-(R)-1 was more potent than (+)-(S)-1 as an inhibitor of alpha 2-mediated platelet aggregation. 相似文献
4.
Hubregtse T Neeleman E Maschmeyer T Sheldon RA Hanefeld U Arends IW 《Journal of inorganic biochemistry》2005,99(5):1264-1267
The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O). 相似文献
5.
Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane. 相似文献
6.
B I Cohen T Kuramoto M A Rothschild E H Mosbach 《The Journal of biological chemistry》1976,251(9):2709-2715
The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates. 相似文献
7.
Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter. 总被引:5,自引:2,他引:3
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule. 相似文献
8.
The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding. 相似文献
9.
It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J. J., & Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin (alpha 2M) normally resulting from reaction of alpha 2M with methylamine. The kinetics of reaction of DNPSCN with alpha 2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of alpha 2M with methylamine [Larsson, L. J., & Bj?rk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS is liberated per mole of free thiol in alpha 2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3(-) can also react with the methylamine-generated thiol groups of alpha 2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3(-) inhibits the conversion of alpha 2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated alpha 2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of alpha 2M.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates. 相似文献
11.
The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple alpha T(GDP) molecules is dependent on [rhodopsin] and when [alpha T] greater than [rhodopsin], the activation of the total alpha T pool may be limited by the rate of dissociation of rhodopsin from the activated alpha T(GTP) species; and (3) under conditions of optimal rhodopsin-alpha T coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event. 相似文献
12.
Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin 总被引:2,自引:0,他引:2
alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Iu M Evdokimov S G Skuridin A T Dembo E V Shtykova V A Kadykov 《Molekuliarnaia biologiia》1979,13(5):1110-1123
Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A).Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml. 相似文献
14.
Toniolo C Formaggio F Tognon S Broxterman QB Kaptein B Huang R Setnicka V Keiderling TA McColl IH Hecht L Barron LD 《Biopolymers》2004,75(1):32-45
We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution. 相似文献
15.
Density function theory calculations of frequency dependent optical rotations ([alpha]omega) for 30 rigid chiral molecules are reported. Calculations have been carried out at the sodium D line frequency, using the augmented double zeta valence quality plus polarization functions (ADZP) basis set and the BP86 nonhybrid and B3LYP hybrid functionals. Gauge-invariant atomic orbitals were used to guarantee origin-independent values of [alpha]D. Comparison between corresponding results obtained with nonhybrid and hybrid functionals as well as with theoretical optical rotations reported in the literature is done. Excited electronic states of three molecules are also discussed in light of circular dichroism spectra and B3LYP and BP86 calculated excitation energies and rotatory strengths. One verifies that the B3LYP/ADZP results are in better agreement with experiment. 相似文献
16.
A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the alpha-tocopherol binding protein (alpha TBP) mediated transfer of alpha-tocopherol. Liposomes containing RRR-[alpha-3H]tocopherol ([alpha-3H]T) were incubated with acceptor membrane at 37 degrees C for 0-45 min in the presence or absence of rat liver cytosol or a dialyzed 30-60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. alpha-Tocopherol transfer activity (alpha TTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one alpha TTA peak (liver mitochondria as acceptor) with an estimated Mr of 39,000. [alpha-3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [alpha-3H]T was transferred intact. alpha TTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [alpha-3H]T followed saturation kinetics. Optimal concentrations gave alpha TTA responses directly proportional to rat liver cytosol concentration. alpha TTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal alpha-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of alpha-tocopherol is needed for transfer. Coefficient of variation of repeated measures of alpha TTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of alpha-tocopherol can be studied quantitatively and can be used to compare liver protein preparations exhibiting transfer activity. 相似文献
17.
Translocation of the alpha subunit of Gi2 from the membrane to the cytosol was studied in mouse mastocytoma P-815 cells. To monitor Gi2 alpha the membrane (300,000 x g pellet) was [32P]ADP-ribosylated with pertussis toxin. Incubation of the [32P]ADP-ribosylated membrane with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a small release (10%) of [32P]ADP-ribosylated Gi2 alpha from the membrane. Whereas cytosol (300,000 x g supernatant) alone had no ability to release the [32P]ADP-ribosylated Gi2 alpha from the membrane, it markedly augmented the release induced by GTP gamma S, about 50% of the total [32P]ADP-ribosylated Gi2 alpha being released by 30 min. The GTP gamma S-induced release and its enhancement by the cytosol were specific for GTP and GTP gamma S. When the cytosol was boiled this promoting activity was lost. The [32P]ADP-ribosylated Gi2 alpha released by the cytosol plus GTP gamma S from the membrane was eluted as a single peak corresponding to a molecular weight of about 100,000 from an Ultrogel AcA 44 column. In contrast, the [32P]ADP-ribosylated Gi2 alpha released by GTP gamma S alone was eluted at the position of Mr = 40,000, but it was eluted at the position of Mr = about 100,000 when it was incubated with the cytosol. Furthermore, Gi2 alpha purified from bovine lung also behaved in a similar way on gel filtration. The addition of thrombin, a stimulant of histamine secretion from mast cells, to mastocytoma cells drastically induced the translocation of Gi2 alpha from the membrane to the cytosol in a pertussis toxin-sensitive manner. These results taken together demonstrate that the cytosol contains some factor(s) that promotes the release of GTP-activated Gi2 alpha from the membrane and that the released Gi2 alpha exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells. 相似文献
18.
Biosynthesis of mammary glycoproteins. Partial characterization of the sequence for the assembly of lipid-linked saccharides 总被引:2,自引:0,他引:2
Incubation of a membrane preparation from the lactating bovine mammary gland with UDP-[3H]GlcNAc, GDP-[14C]Man, and UDP-[3H]Glc results in the biosynthesis of 15 lipid-linked saccharides that differ from one another by a monosaccharide unit. Pulse and chase kinetics indicate that these glycolipids are related to one another as precursor products for the biosynthesis of asparagine-linked glycoproteins of this tissue. [Man-14C]- and [Man-14C, GlcNAc-3H]saccharides were prepared from corresponding glycolipids by mild acid hydrolysis. Following extensive purification by paper and gel filtration chromatography, structural characterization was conducted on tri-, tetra-, penta-, and undecasaccharides via size determination on calibrated columns of Bio-Gel P-2 and P-4, compositional analysis, exo- and endoglycosidase digestions, methylation, Smith degradation, and acetolysis. These structures were identified as: Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)Glc-NAc, Man alpha 1 leads to 3Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc, Man alpha 1 leads to 3(Man alpha 1 leads to 6)Man beta 1 leads to 4(3)Glc NAc beta 1 leads to 4(3)Glc-NAc, and Man alpha 1 leads to 2 Man alpha 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 2Man alpha 1 leads to 6[Man alpha 1 leads to 2Man alpha 1 leads to 3]Man alpha 1 leads to 6)Man beta 1 leads to 4(3)GlcNAc beta 1 leads to 4(3)GlcNAc. 相似文献
19.
Double-helical character of ribonucleic acid from virus-like particles found in Penicillium chrysogenum 总被引:8,自引:5,他引:3
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. RNA was isolated from virus-like particles found in Penicillium chrysogenum and resolved into two fractions by gel filtration through agarose columns. 2. Fraction 1 was excluded and had the following properties: 50.9% G+C [AMP 0.246, UMP 0.246, CMP 0.252, GMP 0.255 (mole fraction)]; mol.wt. about 1.2x10(6) daltons; s(20,w) 12.3S and ;melting' temperature about 100 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.181mol(-1) cm(-1) at 260nm. 3. Properties of fraction 2 include 37.8% G+C [AMP 0.313, UMP 0.312, CMP 0.186, GMP 0.189 (mole fraction)]; mol.wt. about 140000 daltons; s(20,w) 7.3S, T(m) about 85 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate, pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.241mol(-1) cm(-1) at 260nm. 4. The properties of both fractions were consistent with a double-helical conformation. 相似文献
20.
A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献