首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.  相似文献   

2.
ESR spectra have been recorded of one-electron reduced thymine (T.-) trapped in a variety of thymine derivatives and matrices. Most of the data were taken at Q-band microwave frequencies on 12 M LiCl glasses containing oligo(T) varying from 2 to 18 nucleotides in length. A nonlinear least-squares simulation program was used to simulate the ESR spectra, resulting in a parameter set that can be used to generate T.- powder spectra at X- and Q-band frequencies. Although it is not possible to identify a unique set of g and hyperfine tensors, it is possible to establish boundaries for these interactions and propose a parameter set that is likely and reasonable. As the trapping molecule is varied from TdR to oligo(T) to poly(T), there are small changes in the interaction parameters. The changes are not large enough to account for the differences between hfc values determined from DNA fiber data versus hyperfine coupling values measured from monomer systems. The analysis also indicates that, in these systems, T.- does not protonate between 4 and 77 K at neutral pH.  相似文献   

3.
ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra.  相似文献   

4.
Benjwal S  Jayaraman S  Gursky O 《Biochemistry》2005,44(30):10218-10226
High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.  相似文献   

5.
Interaction of micellar complexes apolipoprotein A1--phosphatidyl choline (apoA1--DMPC and apoA1--EPC) with complex components: apoA1 (dansyl-A1) and phosphatydil cholines (DMPC, EPC and spin labelled PC) was studied in the absence of lipoproteins and plasma components. Recombination of the complexes (changes in complex sizes and stoichiometry) was shown to occur in the presence of the complex components. Interaction of lipid-free apoA1 with the complex is a fast process; incorporation of PC molecule takes place more slowly. This recombination is considered to be a kinetikally complicated process, the rate of recombination depending on PC exchange and interconversion.  相似文献   

6.
At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.  相似文献   

7.
Cai Z  Sevilla MD 《Radiation research》2003,159(3):411-419
An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.  相似文献   

8.
The electron spin resonance (ESR) spectra of human and rabbit ferriheme-hemopexin complexes at 30oK show an ESR absorption characterized by gx = 1.60, gy = 2.25 and gz = 2.86, characteristic of low-spin ferriheme-proteins. The low-spin nature of the heme-iron in heme-hemopexin is corroborated by the observation of nuclear hyperfine splitting in M?ssbauer spectra at 4.2oK. The similarity of the ESR spectra of ferriheme-hemopexin with those of low-spin cytochromes which coordinate heme via two histidine residues points to a similar coordination mechanism in hemopexin. In contrast, the ESR spectra of the 1:1 and 2:1 complexes of heme with human serum albumin display signals at g = 6.0 and g = 2.0, characteristic of high-spin ferrihemeproteins.  相似文献   

9.
The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters (Ka approximately 10(5) M-1; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: deltaG=-6.8 kcal/mol; deltaH=-7.9 kcal/mol; deltaS=-3.2 cal/mol per degree and confirmed a transition temperature of about 65 degrees C for albumin. Na, Liland Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity (Azz coupling constant) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.  相似文献   

10.
Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.  相似文献   

12.
For the characterization of the substrate binding site optical and EPR measurements with spin labelled substrates on solubilized and pure cytochrome P-450 were performed. Analogously to the unlabelled derivatives spin labelled n-alkylamines and isocyanides with different chain lengths are type II substrates. The Ks-values evaluated from optical (P-450 = 1.98 . 10(-6) M) and ESR (P-450 = 1.98 . 10(-4) M) measurements are very similar indicating no concentration dependences. Contrary to the unlabelled n-alkylamines the spin labelled compounds show an affinity almost independent of the chain lengths. The SL-substrates with a short distance between the functional group and the NO-group bound to P-450 induce pronounced changes of the ligand field of the heme iron and a large broadening of the signal of the immobilized nitroxide indicating intensive interactions between the unpaired electron of the nitroxide group and the paramagnetic heme iron. Elongation of the alkyl chains results in spectra of the Fe3+ complexes with only slight modification and a remained unbroadened signal of the immobilized nitroxide. The binding of the substrate through their functional groups together with a 1:1 stoichiometry of the P-450 SL-IC-complex give evidence for the same binding site in the near vicinity of the heme iron.  相似文献   

13.
Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.  相似文献   

14.
Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.  相似文献   

15.
Six transition metal ion complexes have been examined for their effects on the cell survival as well as their effectiveness in inducing the broadening of the electron spin resonance (ESR) spectra of nitroxide spin probes. These paramagnetic species are Ni(EDTA), Ni(DTPA), potassium tris(oxalato) chromate (chromium oxalate), K3Fe(CN)6, Cu(DTPA), and NiCl2. At 100 mM concentration, the typical concentration used in cell studies to broaden the extracellular nitroxide ESR signal, only Ni(EDTA) and Ni(DTPA) are found to be non-toxic to Chinese hamster ovary cells. The relative cytotoxicities of the six metal ion complexes are Cu(DTPA) greater than K3Fe(CN)6 greater than NiCl2 greater than chromium oxalate greater than Ni(DTPA) greater than Ni(EDTA). Thus, potassium ferricyanide and NiCl2, two most commonly used paramagnetic broadening agents, are relatively toxic to the cell. In contrast, among the six paramagnetic species tested here, chromium oxalate appears to be the most effective agent at non-toxic concentrations in inducing the broadening of the ESR spectra of both cationic and neutral nitroxide spin probes. By considering both their cytotoxicity and their effectiveness in causing line broadening of the nitroxide ESR spectra, chromium oxalate is a good paramagnetic broadening agent for spin probe studies of intact mammalian cells.  相似文献   

16.
A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.  相似文献   

17.
The nitrosyl derivatives of Annelidae Glossoscolex paulistus hemoglobin (an earth worm erythrocruorin (Ec AGp)) and Aplysia brasiliana myoglobin (Mb Apb) are studied using ESR spectroscopy. These two proteins have a quite similar ESR spectra at 100 K, but a different temperature behaviour. The temperature dependence of the nitrosyl Mb Apb spectrum is in good agreement with the Boltzmann distribution. In the case of nitrosyl-Ec AGp, the results are explained by the existence of two types of spectrum in thermodynamic equilibrium, with delta H = 9.08 kJ/mol, delta S = 47.15 J/mol and T1/2 = 193 K. There is a great similarity of the nitrosyl-Ec AGp spectra with those reported for elephant myoglobin, suggesting the presence of the same heme environment with a glutamine residue in the distal site. The pH dependence of the spectrum of nitrosyl-Mb Apb shows that the affinity of nitrosyl binding is higher at high pH (7.3) than at low pH (4.6). The ESR parameters are the same for these two pH values.  相似文献   

18.
Iron nitrosyl haemoglobin (HbFeNO) gives well defined ESR spectra, and can be detected at room temperature, in contrast with most transition metal complexes of biological importance. This is because the unpaired electron remains strongly localised on the NO ligand. It is of importance because it proves the formation of nitric oxide, which unfortunately cannot be detected directly by ESR spectroscopy. We have studied a range of tissues taken from human liver, colon and stomach tumours which have been directly frozen to 77K and studied at 77K. The results show that formation of HbFeNO is rare in tissue adjacent to tumour tissue (“peripheral tissue”), but is always found in necrotic central regions, if present. However, in several cases, HbFeNO was also detected in tumour tissue which was not necrotic. Two factors contribute to the formation of this complex. One is the presence of “free” NO molecules in the cellular regions, and the other is the presence of deoxyferrohaemoglobin, since neither ferrihaemoglobin nor oxyhaemoglobin react to give this complex. [For systems containing myoglobin these comments include the possibility of the formation of nitrosylmyoglobin, which gives very similar ESR spectra.]  相似文献   

19.
The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.  相似文献   

20.
Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b+ F4/80+ macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号