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1.
We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times. 相似文献
2.
K Maruyama T Yuda A Okamoto S Kojima A Suginaka M Iwatsuru 《Biochimica et biophysica acta》1992,1128(1):44-49
The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors. 相似文献
3.
German A. B. Neklyudov A. D. Ivankin A. N. Berdutina A. V. 《Applied Biochemistry and Microbiology》2002,38(6):517-520
Hydrolysis of lipids from beef fat by pancreatic lipase was studied. The maximum release of free fatty acids was shown to occur at 40°C for the first 3 h of the experiment. After this, transetherification was predominant. The main kinetic parameters were the following: maximum hydrolysis rate, V = 1.25 ± 0.1 mg fat/ml min; Michaelis constant, K
M
H = 100 ± 12 mg fat/ml; constant of substrate inhibition, K
S = 10.0 ± 0.8 mg fat/ml; equilibrium constant, K
P = 277 ± 170 mg fat/ml; and activation energy of beef fat hydrolysis by pancreatic lipase, E
a = 19.1 ± 1.1 kJ/mole. The kinetic method used could be applied to development of the method for biotransformation of poorly assimilated fats into more valuable products. 相似文献
4.
Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes
Dos Santos N Mayer LD Abraham SA Gallagher RC Cox KA Tardi PG Bally MB 《Biochimica et biophysica acta》2002,1561(2):188-201
To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes. 相似文献
5.
C.L. Bashford G.M. Alder L.G. Fulford Y.E. Korchev E. Kovacs A. MacKinnon C. Pederzolli C.A. Pasternak 《The Journal of membrane biology》1996,150(1):37-45
Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus α-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded
liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with α-toxin in the presence of PEG
with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl)diazirine(125I-TID) labels monomeric and—predominantly—hexameric forms of liposome-associated α-toxin; in the absence of PEG little labeling
is apparent. At high concentrations of H+ and Zn2+ but not of Ca2+—all of which inhibit calcein leakage—the distribution of label between hexamer and monomer is perturbed in favor of the latter.
In α-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote
the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence
of nonelectrolytes; they differ only in being more sensitive to ``closure' by voltage (as are pores induced in cells). In
both systems, the stimulation by nonelectrolytes increases with concentration and with molecular mass up to a maximum around
2,000 Da. We conclude (i) that most of the α toxin that becomes associated with liposome or planar lipid bilayers does not
form active pores and (ii) that the properties of α-toxin-induced pores in lipid bilayers can be modulated to resemble those
in cells.
Received: 2 October 1995/Revised: 3 November 1995 相似文献
6.
Manabu Ueno Hyun Seung Ban Kei Nakai Ryu Inomata Yasufumi Kaneda Akira Matsumura Hiroyuki Nakamura 《Bioorganic & medicinal chemistry》2010,18(9):3059-3065
closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 1012 neutrons/cm2). 相似文献
7.
Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a
specific mass transfer area
- Dm
molecular diffusion coefficient (m2/sec)
- e1, e2
charge of the protein and the gel
- k
apparent mass transfer coefficient (s-1)
- ka
global mass transfer coefficient
- f
partition coefficient
- Kp
dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent)
- p
equilibrium concentration of free enzyme, (mg free enzyme/mL solution)
- q
equilibrium concentration of adsorbed enzyme, (mg ads./mL gel)
- qm
maximum adsorption capacity, (mg ads./ml gel)
- Re
particle Reynolds number
- Sh
Sherwood number
- Vg/V
gel volume (mL)/bulk solvent volume (mL)
- Z
dimensionless extent of adsorption
-
Kp/Po , model parameter
-
(/) +1 , model parameter
-
Vg qm / V Po , model parameter 相似文献
8.
Lactosyl- and melibiosyl-phosphatidylethanolamine prepared by reductive animation with sodium cyanoborohydride were incorporated into small unilamellar liposomes. Lactosyland melibiosyl-phosphatidylethanolamine liposomes are aggregated by Ricinus communis agglutinin whereas Banderiaea simplicifolia isolectin I aggregates only melibiosyl-phosphatidylethanolamine liposomes. The association constant (Ka) values of interactions of R. communis agglutinin and glycolipids were 5 × 105 and 1.2 × 105m?1 for lactosyl- and melibiosyl-phosphatidylethanolamine, respectively, whereas the Ka for the interaction of B. simplicifolia isolectin I for melibiosyl-phosphatidylethanolamine was found to be 6 × 105m?1. The rates of aggregation of these liposomes are strikingly influenced by the amount of glycolipid incorporated into them. In vivo studies indicate that lactosyl-phosphatidyl-ethanolamine-containing liposomes are rapidly taken up by hepatic cells due to binding of their β-d-galactopyranosyl residues by the hepatic galactose-binding lectin. 相似文献
9.
Jeff Reeve Lorraine H. Kligman Robert Anderson 《Applied microbiology and biotechnology》1990,33(2):161-166
Summary Isolated lipids from Deinococcus radiodurans were reconstituted at final concentrations of 1 mg/ml into dioleoyl phosphatidyl choline (DOPC) vesicles and assayed for the ability to protect cells of Escherichia coli against killing by UV light (254 nm). Values of D37 (UV dose required to reduce the number of surviving cells to 37% of the original number) were calculated from killing curves. E. coli was afforded the greatest protection with an individual lipid, identified as vitamin MK8 (D37=310 J//m2, compared to D37=67 J/m2 for E. coli irradiated in the presence of DOPC alone). Liposome-mediated protection was dependent on UV254 absorbance and not on turbidity-related light-scattering. BOth vitamin MK8 from D. radiodurans and vitamin K1, which is available commercially, showed a similar degree of UV254-protection for E. coli. The UV-protective properties of vitamin K1 were also investigated on mammalian cells in comparison with other natural lipids and known sunscreens. Survival curves were obtained for mouse fibroblast (L) cells irradiated at UV254 in the absence or presence of DOPC liposomes into which were incorporated various natural lipids or standard sunscreen ingredients, all at final concentrations of 1 mg/ml. Experimentally determined values of D37 were as follows: Vitamin K1, 73 J/m2; \-carotene, 44 J/m2; -tocopherol, 20 J/m2; sulisobenzone, 156 J/m2; p-aminobenzoic acid (PABA), 113 J/m2; benzophenone, 80 J/m2; oxybenzone, 61 J/m2 and DOPC alone. 23 J/m2. Vitamin K1, the most protective lipid tested, was also compared with PABA and oxybenzone (all at concentrations of 20 mg/ml; applied topicall) for its ability to protec Skh-hairless mice from UV254-induced erythema, yielding a UV254 protection factor of 3.5. In addition, vitamin K1 (at 100 mg/ml) was able to provide hairless mice with a small degree of UVB protection, as indicated by an experimentally determined Solar Protection Factor of 1.5–2.0. Although it is concluded that vitamin K is not likely to account for the extraordinarily high degree of UV-resistance of D. radiodurans, vitamin K does show characteristics worthy of its consideration as a UV-screening agent.
Offprint requests to: R. Anderson 相似文献
10.
Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated. 相似文献
11.
《Journal of liposome research》2013,23(1):215-223
AbstractToxicity and biodistribution of negatively charged liposomes of the main phospholipid (MPL) from the archaebacterium Thermoplasma acidophilum were tested in mice. MPL liposomes with a diameter of 160–220 nm were prepared by extrusion through polycarbonate filters, or by means of a French pressure cell and screened for central nervous system effects after intraperitoneal (i.p.) injection of 4–324 mg of liposomes per kg body weight in NMRI-mice. Besides increased behavioural activity no pharmacological or toxic effects were detected. No alterations were seen in the morphology of the tissues analyzed. Longterm toxicity after life-long oral application of 30 mg MPL per kg body weight per day starting at the age of 10 weeks was tested in immunosuppressed NMRI-mice. Again, there were no toxic effects on survival. Biodistribution of MPL liposomes labeled with 111In-diethylenetriaminepentaacetic acid stearylamide was examined 15 min and 2.5 h after intravenous injection into ICR-mice. The liposomes were rapidly cleared from the circulation and the majority accumulated in the liver, followed by the spleen. 相似文献
12.
Peptides targeting the human neonatal Fc receptor (FcRn) were conjugated to poly(ethylene glycol) (PEG) polymers to study their effect on inhibition of the IgG:FcRn protein-protein interaction both in vitro and in mice. Both linear (5-40kDa) and branched (20, 40kDa) PEG aldehydes were conjugated to an amine-containing linker of a homodimeric anti-FcRn peptide using reductive alkylation chemistry. It was found that conjugation of PEG to the peptide compromised the in vitro activity, with larger and branched PEGs causing the most dramatic losses in activity. The conjugates were evaluated in transgenic mice for their ability to accelerate the catabolism of human IgG. Optimal pharmacodynamic properties were observed with PEG-peptide conjugates that contained 20-40kDa linear PEGs and a 20kDa branched PEG. The optimal PEG-peptide conjugates were more effective in vivo than the unconjugated peptide control on a mole:mole and mg/kg basis, and represent potential new longer-acting peptide therapeutics for the treatment of humorally-mediated autoimmune disease. 相似文献
13.
Development and characterization of magnetic cationic liposomes for targeting tumor microvasculature
Suman Dandamudi 《生物化学与生物物理学报:生物膜》2007,1768(3):427-438
Cationic liposomes preferentially target tumor vasculature compared to vessels in normal tissues. The distribution of cationic liposomes along vascular networks is, however, patchy and heterogeneous. To target vessels more uniformly we combined the electrostatic properties of cationic liposomes with the strength of an external magnet. We report part I of development. We evaluated bilayer physical properties of our preparations. We investigated interaction of liposomes with target cells including the role of PEG (polyethylene-glycol), and determined whether magnetic cationic liposomes can respond to an external magnetic field. The inclusion of relatively high concentration of MAG-C (magnetite) at 2.5 mg/ml significantly increased the size of cationic liposomes from 105 ± 26.64 to 267 ± 27.43 nm and reduced the zeta potential from 64.55 ± 16.68 to 39.82 ± 5.26 mv. The phase transition temperature of cationic liposomes (49.97 ± 1.34 °C) reduced with inclusion of MAG-C (46.05 ± 0.21 °C). MAG-C cationic liposomes were internalized by melanoma (B16-F10 and HTB-72) and dermal endothelial (HMVEC-d) cells. PEG partially shielded cationic charge potential of MAG-C cationic liposomes, reduced their ability to interact with target cells in vitro, and uptake by major RES organs. Finally, application of external magnet enhanced tumor retention of magnetic cationic liposomes. 相似文献
14.
Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations 总被引:1,自引:0,他引:1
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Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl2. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl2 the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K+ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl2 and probably played a key role in determining growth capacity. (b) Internal concentrations of Na+, Ca2+, or Cl− could not be directly correlated with growth. (C) Internal Mg2+ concentration could be significant only in the presence of high external Ca2+ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca2+)/(Na+)2 concentration is crucial for growth. Ratios above 0.5 × 10−4 per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca2+)/(Na+)2 ratio and the absolute external concentration of NaCl. However, a correlation between internal K+ concentration and growth capacity seemed independent of external NaCl concentration. 相似文献
15.
《Journal of Fermentation and Bioengineering》1989,67(4):300-302
2, 6-Dichlorophenolindophenol (DCIP)-dependent polyethylene glycol (PEG) dehydrogenase activity was found in the particulate fractions of cell-free extracts prepared from PEG-utilizing bacteria (Pseudomonas and Flavobacterium species). This result suggested that PEG dehydrogenase is linked to the respiratory chain of each bacterium and that the enzyme plays a major role in the aerobic metabolism of PEG. Enzyme activities were strongly inhibited by 1, 4-benzoquinone. No metal ion was indispensable for the enzyme activities. Enzyme activities of PEG-utilizing bacteria were induced by PEG except for the activity of PEG 4000-utilizing Flavobacterium sp. no. 203 which had a constitutive enzyme. Although PEG-utilizing bacteria had different growth substrate specificities toward PEGs 200–20,000, their PEG dehydrogenases oxidized the same molecular wt. range of PEGs (dimer-20,000). Cell-free extracts of PEG 400-, 1000- or 4000-utilizing bacteria oxidized PEG 6000 and 20,000 though these bigger PEGs could not be utilized as the sole carbon and energy sources by the bacteria. Methanol, ethylene glycol and glycerol were not or only barely dehydrogenated by all the enzyme preparations. 相似文献
16.
G. S. Dotsenko M. V. Semenova O. A. Sinitsyna S. W. A. Hinz J. Wery I. N. Zorov E. G. Kondratieva A. P. Sinitsyn 《Biochemistry. Biokhimii?a》2012,77(11):1303-1311
Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K m and k cat values are 0.4 mM and 15 sec?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K m and k cat values are 1.3 mg/ml and 67 sec?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K m and k cat values are 0.08 mM and 35 sec?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively. 相似文献
17.
We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (KD) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, KD(bulk) = 1.7 ± 0.2 nM vs. 2.9 ± 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, KD = 3.6 ± 1.1 nM vs. 2.0 ± 0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the KD value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 ± 1.1 μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (KD = 21 ± 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands. 相似文献
18.
Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage 总被引:1,自引:0,他引:1
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A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (Km) and Vmax for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively. 相似文献
19.
A new method for immobilizing protein on the surface of liposomes is described. Inclusion of stearylamide in the lipid composition of vesicles resulted In liposomes that could be ‘activated’ by diazotization with NaNO2/HCl, and subsequently coupled with protein. Using this method egg albumin / μmol phospholipid has been coupled to multilamellar vesicles composed of phosphatidylcholine, cholesterol, and stearylamide in a molar ratio of 15:7.5:1.1. Furthermore, when the immunologic response of mice to egg albumin that was encapsulated in, nonspecifically adsorbed, or covalently linked to liposomes was investigated, only the covalent protein-liposome conjugates elicited pronounced and sustained elevations in antibody titers. These results suggest that the immunoadjuvant effects of liposomes can be maximized by covalently linking protein antigens to their surface. 相似文献
20.
Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces. 相似文献