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1.
Studies on the response of cholesterol biogenesis in feeding in rats: evidence against the existence of diurnal rhythms. 总被引:3,自引:3,他引:0
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1. The biosynthesis of cholesterol was studied, by using various precursors, in rats subjected to several dietary regimes. 2. The use of 3H2O as a substrate to demonstrate differences in cholesterogenesis under various conditions was validated by using rats fed on cholesterol or cholestyramine. Cholesterol feeding resulted in decreased cholesterogenesis, whereas cholestyramine caused an increase. 3. With acetate as precursor, the biosynthesis of both digitonin-precipitable sterol and fatty acids was increased in vitro in response to a meal. 4. In rats fed ad libitum, hepatic cholesterogenesis was increased at midnight relative to mid-morning as measured by using acetate precursor in vitro. However, no such difference was found by using 3H2O in vivo. 5. The lipogenic response was measured in meal-fed rats by using 3H2O or octanoate in vivo. In contrast with findings with acetate in vitro, no postprandial stimulation of cholesterogenesis was seen with either 3H2O or octanoate as precursor, whereas fatty acid biosynthesis from either substrate was increased. 6. These findings are discussed with respect to current theories about the circadian rhythm of cholesterogenesis. Such theories are based on experiments using isolated enzyme measurements or non-physiological precursors such as acetate. 7. It is considered that results obtained with 3H2O give an accurate representation of cholesterogenesis under various conditions, and it is therefore suggested that hepatic cholesterogenesis in rats is not subjected to the same degree of diurnal rhythm as has previously been believed. 相似文献
2.
dos Anjos Ferreira AL Yeum KJ Russell RM Krinsky NI Tang G 《The Journal of nutritional biochemistry》2004,15(8):493-502
Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD+, KCI, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of 2H10 lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C18H24O2 or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z =272 and (2) 3,4-dehydro-5,6-dihydro-15-apo-lycopenal (C20H28O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-l-al) with lambdamax= 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-ene-5,8-lycopenal-furanoxide (C37H50O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C40H56O2) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z =568; (5) lycopene-5,8-furanoxide isomer (I) (C40H56O2) with lambdamax = 410 nm, 440 nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C40H56O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C40H54O2) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase. 相似文献
3.
Ferreira AL Yeum KJ Russell RM Krinsky NI Tang G 《The Journal of nutritional biochemistry》2003,14(9):531-540
Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD(+), KCl, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of (2)H(10) lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C(18)H(24)O(2) or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z = 272 and (2) 3,4-dehydro-5,6-dihydro-15,15'-apo-lycopenal (C(20)H(28)O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-1-al) with lambdamax = 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-apo-5,8-lycopenal-furanoxide (C(37)H(50)O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C(40)H(56)O(2)) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z = 568; (5) lycopene-5,8-furanoxide isomer (I) (C(40)H(56)O) with lambdamax = 410 nm, 440nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C(40)H(56)O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C(40)H(54)O(2)) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase. 相似文献
4.
Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide-Induced Oxidative Stress in Nerve Terminals 总被引:8,自引:0,他引:8
Mitochondrial membrane potential (delta psi(m)) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H2O2 (0.1-1 mM) caused only a minor decrease in delta psi(m). When complex I of the respiratory chain was inhibited by rotenone (2 microM), delta psi(m) was unaltered, but on subsequent addition of H2O2, delta psi(m) started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in delta psi(m) when added after oligomycin (10 microM), an inhibitor of F0F1-ATPase. H2O2 (0.1 or 0.5 mM) inhibited alpha-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine delta psi(m) in the presence of H2O2: (a) The NADH level reduced owing to inhibition of alpha-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of delta psi(m) when the F0F1-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of delta psi(m) by F0F1-ATPase. The results indicate that to maintain delta psi(m) in the nerve terminal during H2O2-induced oxidative stress, both complex I and F0F1-ATPase must be functional. Collapse of delta psi(m) could be a critical event in neuronal injury in ischemia or Parkinson's disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired. 相似文献
5.
Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events. 相似文献
6.
We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive. 相似文献
7.
We have reported recently (Chinopoulos et al., 1999 J. Neurochem. 73, 220 228) that mitochondrial membrane potential (delta(psi)m) in isolated nerve terminals is markedly reduced by H2O2 in the absence of F0F1-ATPase working as a proton pump. Here we demonstrate that delta(psi)m reduced by H2O2 (0.5 mM) in the presence of oligomycin (10 mM), an inhibitor of the F0F1-ATPase, was able to recover by the addition of catalase (2000 U). Similarly, a decrease in the NAD(P)H level due to H2O2 can be reversed by catalase. In addition, H2O2 decreased the ATP level and the [ATP]:[ADP] ratio measured in the presence of oligomycin reflecting an inhibition of glycolysis by H2O2, but this effect was not reversible. The effect of H2O2 on delta(psi)m in the presence of the complex I inhibitor, rotenone, was also unaltered by addition of catalase. These results provide circumstantial evidence for a relationship between the decreased NAD(P)H level and the inability of mitochondria to maintain delta(psi)m during oxidative stress. 相似文献
8.
The catalase reaction has been studied in detail by using myoglobin (Mb) mutants. Compound I of Mb mutants (Mb-I), a ferryl species (Fe(IV)=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid. Upon the addition of H2O2 to an Mb-I solution, Mb-I is reduced back to the ferric state without forming any intermediates. This indicates that Mb-I is capable of performing two-electron oxidation of H2O2 (catalatic reaction). Gas chromatography-mass spectroscopy analysis of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e = 36) and 16O2 (m/e = 32) but not 16O18O (m/e = 34). This implies that O2 is formed by two-electron oxidation of H2O2 without breaking the O-O bond. Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus catalase and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction. In contrast, other Mb mutants such as H64X (X is Ala, Ser, and Asp) and L29H/H64L Mb oxidize H2O2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10-29, due to a lack of the general acid-base catalyst. 相似文献
9.
Xiang QX Zhang J Liu PY Xia CQ Zhou ZY Xie RG Yu XQ 《Journal of inorganic biochemistry》2005,99(8):1661-1669
The syntheses, characteristics of dinuclear macrocyclic polyamine zinc complexes and their interaction with plasmid DNA are reported. The two cyclen (1,4,7,10-tetraazacyclododecane) moieties are bridged by rigid and flexible linkages. The crystal structures of Zn2C27H43N8O15Cl4 [5c.(ClO4)3.2H2O] and Zn2C30H43N10O13Cl3 [5e.(ClO4)3.H2O] have been determined. The complexes crystallize in the monoclinic space group C2/c and P2(1)/c with the following unit cell parameters: 5c.(ClO4)3.2H2O: a=32.568(4)A, b=14.8593(17)A, c=19.443(2)A, alpha=90.00 degrees , beta=119.435(4) degrees , gamma=90.00 degrees , Dc=1.551 mg/m3, FW=956.71, F(000)=3932; 5e.(ClO4)3.H2O: a=15.807(2)A, b=16.756(2)A, c=16.161(2)A, alpha=90.00 degrees , beta=97.062(4) degrees , gamma=90.00 degrees , Dc=1.546 mg/m3, FW=988.83, F(000)=2032. The distance between the two Zn(II) ions is about 4.0 A. The structures show that two zinc ions can synergistically interact with the substrate DNA. With this novel structural characteristics, the dinuclear macrocyclic polyamine Zn(II) complexes via the synergetic effect between the two zinc ions can catalyze the cleavage of plasmid DNA (pUC18) with unprecedented speed at physiological conditions. 相似文献
10.
Despite catalyzing the same reaction (2 H2O2-->2 H2O+O2) heme-containing monofunctional catalases and bifunctional catalase-peroxidases (KatGs) do not share sequence or structural similarities raising the question of whether or not the reaction pathways are similar or different. The production of dioxygen from hydrogen peroxide by monofunctional catalases has been shown to be a two-step process involving the redox intermediate compound I which oxidizes H2O2 directly to O2. In order to investigate the origin of O2 released in KatG mediated H2O2 degradation we performed a gas chromatography-mass spectrometry investigation of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing KatGs from Mycobacterium tuberculosis and Synechocystis PCC 6803. The GC-MS analysis clearly demonstrated the formation of (18)O2 (m/e = 36) and (16)O2 (m/e = 32) but not (16)O(18)O (m/e = 34) in the pH range 5.6-8.5 implying that O2 is formed by two-electron oxidation without breaking the O-O bond. Also active site variants of Synechocystis KatG with very low catalase but normal or even enhanced peroxidase activity (D152S, H123E, W122F, Y249F and R439A) are shown to oxidize H2O2 by a non-scrambling mechanism. The results are discussed with respect to the catalatic mechanism of KatG. 相似文献
11.
13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Enzymatic reaction intermediates of horseradish peroxidase, compounds I and II, were studied by high-resolution nuclear magnetic resonance spectroscopy at 220 MHz. The heme peripheral proton peaks were successfully obtained in the downfield region of 50 to 80 ppm from 4,4-dimethyl-4-silapentane-5-sulfonate for compound I and of 10 to 20 ppm for compound II at pH 9.2. This indicates that no isoporphyrin appears in the catalytic cycle of the enzyme. Temperature dependences of the spectra also were determined for these compounds between 7 and 32 degrees C. With increasing temperature, all the peaks in the downfield region for compound I shifted upfield, obeying the Curie law. These results suggest that the Fe atoms in compounds I and II are in ferryl high- and low-spin states, respectively. The spectrum was also observed in solutions of horse metmyoglobin to which hydrogen peroxide (H2O2) was added. The electron formulations of the hemes in their spectra. Evidence was found against a pi-cation radical on the heme ring as a source of the oxidizing equivalent in compound I. 相似文献
13.
D S?awińska J S?awiński 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》1987,1(3):171-175
Ultraweak luminescence in the spectral region 300-660 nm is generated in the enzymatic (tyrosinase EC.1.14.18.1) and autooxidative polymerization of L-DOPA, 5-S-cysteinyl-DOPA, and L-DOPA + cysteine to eumelanins and pheomelanins, respectively. Using sensitive calibrated single photon counting equipment, the photon emission intensity I and quantum yield phi have been measured: I = 10-100 h nu/s cm3, phi less than or equal to 10(-13) for enzymatic reactions, and I = 500-3000 h nu/s cm3, phi greater than or equal to 5 x 10(-12) for autooxidative ones. 5-S-cysteinyl-DOPA and cysteine exhibit diminished I and phi-values relative to DOPA. Tests with chemiluminogenic probes-luminol and lucigenin, SOD, catalase, peroxidase, H2O2, and spectrophotometric measurements indicate that photon emission is associated with degradative oxidations of melanin subunits by means of active oxygen species as H2O2 and O2. 相似文献
14.
Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium 总被引:23,自引:0,他引:23
Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs. 相似文献
15.
J H Hochman Y Shimizu R DeMars M Edidin 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(7):2322-2329
We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m. 相似文献
16.
P G Schmidt M W Holladay F G Salituro D H Rich 《Biochemical and biophysical research communications》1985,129(2):597-602
The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3-13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-al anyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H216O and H218O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms. 相似文献
17.
Yang L Xie D Xu Y Wang Y Zhang S Weng S Zhao K Wu J 《Journal of inorganic biochemistry》2005,99(5):1090-1097
A single crystal of a coordinated complex of neutral erythritol (C4H10O4,E) with a neodymium ion, NdE(II), was synthesized and studied using FT-IR and X-ray diffraction analysis. In NdE(II) (NdCl3.2.5C4H10O4.C2H5OH) the Nd3+ coordinates with one chloride ion and eight OH groups from three erythritol molecules. There are two neodymium centers linked by one erythritol molecule with same coordination structure in the molecule. Two erythritol molecules provide 1,3,4-hydroxyl groups to coordinate with a neodymium ion; another erythritol molecule coordinates to two Nd ions via its 1,2-hydroxyl groups and 3,4-hydroxyl groups, respectively. The OH groups of erythritol act as ligand to coordinate to neodymium ions, and OH groups of erythritol form hydrogen bond networks that link chain and layer together to build three-dimensional structures. The ratio of metal to ligand is 1:2.5. The structure of NdE(II) is more complicated than the previously reported NdE(I), which is NdCl3.C4H10O4.6H2O; in NdE(I), Nd3+ is coordinated to four hydroxyl groups from two erythritol molecules, four water molecules and one chloride ion. The results indicate the complexity of metal-sugar interaction. 相似文献
18.
A microtechnique for dialysis of small volume solutions with quantitative recoveries 总被引:2,自引:0,他引:2
C M Overall 《Analytical biochemistry》1987,165(1):208-214
An improved microtechnique designed for dialysis of solutions with volumes ranging from less than 10 microliter up to approximately 600 microliter is described. Samples, dispensed in Microfuge tubes, are dialyzed in situ across dialysis membrane secured over the tube opening by a perforated Microfuge tube cap. The retentate is efficiently recovered by centrifugation at 10,000g for 10 s. Fifty percent escape (E50) times of [14C]glycine from 25-microliter solutions of soybean trypsin inhibitor (0.1, 1.0, 4.0 mg/ml) in 0.2 M NaCl were approximately 19 min. The E50 times of 3H2O increased in a linear fashion from 2.7 min for 25-microliter samples to 75 min for 600-microliter samples of H2O (pH 7.0, 4 degrees C). The mean permeability coefficient (P) of the dialysis membrane to 3H2O during microdialysis, calculated as 3.0 X 10(-4) cm/s, was similar to membrane permeability coefficients reported for dialysis by conventional methods. Quantitative recoveries (greater than approximately 90%) of [14C]glycine-labeled type I collagen, [methyl-14C]antithrombin III, and [32P]DNA were achieved after microdialysis. 相似文献
19.
The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses. 相似文献
20.
L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen. 总被引:8,自引:0,他引:8
N S Kwon C F Nathan C Gilker O W Griffith D E Matthews D J Stuehr 《The Journal of biological chemistry》1990,265(23):13442-13445
Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water. 相似文献