共查询到20条相似文献,搜索用时 31 毫秒
1.
Patricia M. Hinkle David G. Lewis 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,541(3):347-359
Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C. 相似文献
2.
Suzanne E. Patton Daniel M. Lapadula Mohamed B. Abou-Donia 《Neurochemistry international》1985,7(1):111-123
The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 μM [γ-32P]ATP, 10 mM MgCl2, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35–75 μg/100 μl for cytosolic proteins and 21–125 μg/200 μl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing. 相似文献
3.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,721(1):141-145
A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 μg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10 000 and centrifuged at 4500 g for 2 h at 4°C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-μl insert and 100 μl was injected into an LC system. The chromatographic system used a C18 reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)–methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 μg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively. 相似文献
4.
A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll? gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg2+ and Ca2+ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg2+ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane. 相似文献
5.
—Highly purified fractions of synaptic vesicles were prepared from rat cerebrum or cerebral cortex by density gradient centrifugation. Treatment of synaptic vesicle fractions by autoincubation, freeze-thawing and sonication in an isotonic alkaline-salt medium or in 0·1-0·3% (v/v) Triton X-100 released increasing quantities of synaptic vesicle protein and phospholipid into solution. When the soluble synaptic vesicle proteins were extracted with 0·1% (v/v) Triton X-100, the insoluble residue consisted mostly of 5–8 nm-thick membranes resembling the limiting membranes of intact synaptic vesicles. This finding, together with other considerations, suggested that the soluble proteins and accompanying phospholipids originated from the interior of the synaptic vesicles. A 0·3% (v/v) Triton X-100 extract of synaptic vesicle was fractionated by ultracentrifugal flotation and dialysis into three lipoprotein fractions: a low density lipoprotein (d < 1·21 g/ml), a high density lipoprotein (d = 1·21–1·35 g/ml) and a very high density lipoprotein (d > 1·35 g/ml). The phospholipid contents of the low, high and very high density lipoprotein fractions were 0·74, 0·38 and 0·20 mg/mg of protein, respectively. All three apolipoproteins had a high ratio of acidic to basic, and of polar to nonpolar, amino acids, and were rich in glycine, alanine and serine. Polyacrylamide gel electrophoresis of the alkaline-salt and Triton X-100 extracts of synaptic vesicles at pH 8·8 resolved a single anionic component which stained for protein, lipid (Sudan black B; iodine) and anionic groups (acridine orange). Polyacrylamide gel electrophoresis of synaptic vesicle extracts at pH 2·7 in 5 m urea and 0·25% (v/v) Triton X-100 resolved about 20 protein components. However, the protein profiles of electropherograms of the Triton X-100 and alkaline-salt extracts differed in certain respects, suggesting that these media to some extent solubilized different proteins. However, most of the protein bands in electropherograms of the Triton X-100 and alkaline-salt extracts also stained for lipid and anionic groups. In addition, two lipoprotein components in the alkaline-salt extract and four in the Triton X-100 extract contained carbohydrate. Isoelectric focusing of synaptic vesicle extracts resolved 6–8 protein fractions. The major fraction in Triton X-100 and alkaline-salt extracts had an apparent isoelectric point of approximately 4·2 and contained 0·24 mg of phospholipid per mg of protein. Soluble synaptic vesicle proteins released by incubating, freeze-thawing and sonicating in the alkaline-salt medium, and protein fractions of the latter obtained by electrofocusing had an absorption maximum of 260–265 nm which was enhanced in a cold 0·5 n perchloric acid extract, an observation suggesting the presence of a bound nucleotide. These findings demonstrate that rat brain synaptic vesicles contain a heterogenous array of soluble acidic lipoproteins which vary in buoyant density, lipid content, amino acid and carbohydrate composition and electrophoretic mobility in polyacrylamide gels. These acidic lipoproteins apparently comprise the bulk of the macromolecular contents of synaptic vesicles and probably serve as ‘carrier’ proteins for the binding and sequestration of the neurotransmitters. 相似文献
6.
Large (0.5–1.0 μm) cytoskeleton-free vesicles were obtained, by ‘budding’, from fresh human and rabbit erythrocytes incubated at 45°C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0–11.0 and temperature range 4–50°C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes. 相似文献
7.
Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors 总被引:1,自引:0,他引:1
The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m2/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores. 相似文献
8.
James T. Russell 《Analytical biochemistry》1981,113(2):229-238
Two different density gradients are described for the isolation of highly purified fractions of neurosecretory vesicles in isoosmotic solutions (300 mosm/kg) from bovine neurohypophyses. The techniques involve differential centrifugation of neural lobe homogenates followed by density gradient centrifugation on metrizamide-sucrose or Percoll-sucrose gradients. The purified fraction contained 44 and 65 μg vasopressin/mg protein, respectively. Neurosecretory vesicles thus isolated were only slightly contaminated with other subcellular organelles, e.g., mitochondria and lysosomes. These vesicles were highly stable in isotonic sucrose solutions (pH 7.5 and 5.5) even at 37°C for at least 2 h, retaining more than 90% of their hormonal content. 相似文献
9.
Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages 总被引:15,自引:0,他引:15
Rabbit alveolar macrophages internalize α-macroglobulin 125I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · 125I-T at 37°C than at 0°C. At 0°C cell-bound αM · 125I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · 125I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · 125I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · 125I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM125I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization. 相似文献
10.
A. J. Sophianopoulos J. A. Sophianopoulos 《Preparative biochemistry & biotechnology》2013,43(4):413-435
Abstract Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6–8°C for 16–18 hr at pH 3.0–3.2 in 1 M MaCl, 0.08 M glycine and 3 mM each Ca+2 and Mn+2, heat treating at 45°C for 2 hr and centrifuqing. The supernatant was neutralized to pH 5 and stored in the cold. The overall yield was 70–80% Some of the properties of Con A at pH 5 are: The absorption coefficient of a 1 g/dl solution is 13.7 at 280 nm; the mean residue elliptic-ity at 224.5 nm is ?9,300° to ?9,800°; by sedimentation equilibrium, its molecular weight is 53,000 between pH 3.0 and pH 5.2. Con A solutions standing at room temperature at pH 7 for ten days lose through precipitation only 5–8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCI. In the solution conditions of SDS and urea-SDS gels, Con A not only unfolds slowly and incompletely, but it also forms high molecular weight aggregates. Thus, electrophoresis of Con A in such gels is unsuitable for tests of homogeneity. However, as judged by sedimentation equilibrium in 6.5 M quanidine at pH 8.1, purified Con A was monodisperse. 相似文献
11.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(1):210-224
Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0°C (pH 7.4) and 20°C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20°C within 2 min with 50–155 and 5.6–17 μM 3 2Pi, respectively, and at 0°C within 75 s with 300–600 and 33–66 μM 3 2Pi, respectively. Maximal phosphorylation yields 146 pmol 3 2P · mg−1 protein. Without ouabain (20°C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached.Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations.Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15–30 s at 0°C and pH 7.4 at concentrations of [γ-3 2P] ATP of 5–32 and 0.5–3.5 μM, respectively. The maximal phosphorylation level is 149 pmol 32P · mg−1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate one site per active enzyme subunit of 135 000 molecular weight.From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0°C and 20°C standard free-energy changes of −5.4 and −6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol · oK. this charactrizes the reaction as a process driven by an entropy change. p ]The intermediate formed by phosphorylation with pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range 0f 4–7. The non-solubilized preparation has optimal stability at ph 2–4, the level of which is equal to that of detergent-solubilized intermediate.Sodium dodecyl gel electrophoreses of the microsomes at pH 3, the following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000-106 000) carries the radioactive label. This protein has the same molecule weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+. 相似文献
12.
S.A. Furlan A.L.S. Schneider R. Merkle M.F. Carvalho‐Jonas R. Jonas 《Engineering in Life Science》2001,21(1):57-64
The pH, temperature and inoculum ratio for the production of β‐galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar‐cane molasses (100 g/l) in a lactose‐free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by‐product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose‐free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β‐galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7. 相似文献
13.
Gas chromatographic assay of total and O-acetyl groups in bacterial lipopolysaccharides 总被引:2,自引:0,他引:2
An improved gas chromatographic method for the determination of total, i.e., amide- and ester-linked, acetyl groups in hydrolysates of bacterial lipopolysaccharides has been developed. After hydrolysis with 0.2 n HCl overnight at 100°C and adjustment of the hydrolysate to pH 3–4, 2 μl of the samples contalning 0–6 μg of acetic and 1–2 μg of propionic acid are directly injected into a gas chromatograph fitted with a 1.5-m glass column packed with Porapak QS. O-Acetyl groups can selectively be determined after hydrolysis with 0.05 n NaOH for 3–4 hr at room temperature and adjustment of the sample to pH 3–4. N-Acetyl groups can be calculated as the difference between total and O-acetyl. The above-mentioned phase allows quantitation of short-chain volatile fatty acids (S-C VFAs) up to n-valeric acid in a range of 0–6.0 μmol using an appropriate internal standard. 相似文献
14.
Transport of long-chain fatty acids into rat adipocytes was previously shown to be inhibited by the reactive derivative sulfosuccinimidyl
oleate consequent to its binding to a membrane protein FAT, which is homologous to CD36. In this report, the ability of the
purified protein to bind native fatty acids was investigated. CD36 was isolated from rat adipocytes by phase partitioning
into Triton X-114 followed by chromatography on DEAE and then on wheat germ agglutinin. Fatty acid binding was determined
by incubating CD36, solubilized in buffer containing 0.1 Triton X-100, with fatty acids at 37°C, and then by adsorbing the
unbound ligand with Lipidex 1,000 at 0°C. Bovine serum albumin was used as a positive control and gelatin, a protein that
does not bind fatty acids, as a negative control. Measurements with albumin yielded reproducible binding values which were
not altered by the presence of 0.1% Triton X-100. Under the same conditions, gelatin yielded reproducibly negative measurements
that did not differ significantly from zero.
CD36 bound various long-chain fatty acids at low ligand to protein ratios. Warming the protein-FA-Lipidex mixture to 37°C
removed the FA off the protein. Thus, binding was reversible and distinct from the palmitoylation of the protein known to
occur on an extracellular domain. Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid
binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment
between residues 127 and 279.
Received: 17 January 1996/Revised: 8 May 1996 相似文献
15.
Pathway of vesicular stomatitis virus entry leading to infection 总被引:67,自引:0,他引:67
The entry of vesicular stomatitis virus into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 °C, viruses bound to the cell surface but were not internalized. Binding was very dependent on pH. More than ten times more virus bound at pH 6.5 than at higher pH values. At the optimal pH, binding failed to reach equilibrium after more than two hours. The proportion of virus bound was irreproducible and low, relative to the binding of other enveloped viruses. Over 90% of the bound viruses were removed by proteases. When cells with pre-bound virus were warmed to 37 °C, a proportion of the bound virus became protease-resistant with a half-time of about 30 minutes. After a brief lag period, degraded viral material was released into the medium. The protease-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride blocked the infection and slightly reduced the degradation of viral protein.When the entry process was observed by electron microscopy, viruses were seen bound to the cell surface at 0 °C and, after warming at 37 °C, within coated pits, coated vesicles and larger, smooth-surfaced vesicles. No fusion of the virus with the plasma membrane was observed at pH 7.4.When pre-bound virus was incubated at a pH below 6 for 30 seconds at 37 °C, about 40 to 50% of the pre-bound virus became protease-resistant. On the basis of this result and previously published experiments (White et al., 1981), it was concluded that vesicular stomatitis virus fuses to the MDCK cell plasma membrane at low pH.These experiments suggest that vesicular stomatitis virus enters MDCK cells by endocytosis in coated pits and coated vesicles, and is transported to the lysosome where the low pH triggers a fusion reaction ultimately leading to the transfer of the genome into the cytoplasm. The entry pathway of vesicular stomatitis virus thus resembles that described earlier for both Semliki Forest virus and fowl plague virus. 相似文献
16.
《Journal of liposome research》2013,23(2):1049-1061
AbstractLipid vesicles may be safely and efficiently loaded with therapeutic dose levels of the beta emitter yttrium-90 (90Y) by using the ability of the cation ionophore A23187 to transport yttrium across the lipid bilayer where it is chelated on the vesicle interior by diethylenetriamine pentaacetic acid (DTPA). For 100 nm diameter vesicles composed of diplamitoylphosphatidylcholine (DPPC) and cholesterol (Choi), DPPC/Chol (1:1), containing 15 mM DTPA with 40 nmoles of external yttrium, total uptake was > 95% of added yttrium within 5 min at 50° using 0.4 ng of ionophore per nmole of lipid. Background binding in these neutral vesicles accounts for less than 0.1% of the yttrium associated with the vesicles. Important operational parameters were the amount of ionophore (> 0.2 μg of ionophore per μmole of lipid was required) and also the temperature (for DPPC/Chol (1:1) vesicles uptake at 40° was essentially background but was > 95% at 50°). The presence of the polymer polyethylene glycol (PEG) on the membrane surface had no effect upon yttrium uptake. Once entrapped, vesicles did not leak any contents for several days at room temperature. 相似文献
17.
J M Brewer 《Archives of biochemistry and biophysics》1974,164(1):322-325
Bakers yeast enolase A binds 2 moles of magnesium with a total enthalpy of +11,000 ± 1,100 cal/mol of protein at 25 °C in 0.05 ionic strength Tris buffer, pH 7.8. Measurements of the pH of unbuffered solutions of enolase indicate that at 0.05 ionic strength, 2 moles of protons are released per 2 moles of metal bound. The binding of magnesium to yeast enolase is consequently produced by a favorable entropy change. The enthalpies of binding observed in Tris buffer appear to be different at 0–1 and 1–2 moles of metal per mole of protein, suggesting a difference in binding sites. 相似文献
18.
The powerful muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) specifically binds to homogenates of Drosophila melanogaster head at a level of 65 ± 6 fmol/mg protein, with an apparent dissociation constant of 0.15–0.7 nM. The half-life of the ligand-receptor complex at 25°C is 30–40 min. Binding is inhibited by low concentrations of muscarinic ligands but not by low concentrations of nicotinic ligands, anticholinesterases or non-cholinergic drugs. Binding-sites are membrane bound and are inactivated by trypsin and by Triton X-100. Part of the activity (<20%) is released into a high speed supernatant by 2 M-NaCI. The gene coding for the putative muscarinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase 相似文献
19.
The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane-enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal-NaF-epine-phrine-, and glucagon-stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8–17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1–5 mM mercaptoethanol, 1 mM MgCl2, and 0.1% Triton X-305. The detergent to membrane protein ratio used in these studies was 3:1. Criteria for solubilization included lack of sedimentation at 100,000 × g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cylcase activity in Sephadex G-200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X-305. The solubilized enzyme was stimulated 5-fold by NaF, 7-fold by glucagon, and 20-fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10-fold, 3,4-fold, and 4-fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at ?60° C. 相似文献
20.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,686(1):19-26
High-performance liquid chromatographic procedures have been developed for the measurement of meropenem in serum. The separation was performed on an Ultrasphere XL-ODS analytical column (75×4.6 mm I.D.). The mobile phase consisted of 10.53 mmol/l ammonium acetate-acetonitrile (95:5, v/v) (pH 4). The UV detection was at 298 nm. The quantitation limit both in serum and water was 0.25 μg/ml. The method was validated in serum and aqueous solution over the concentration range 0.25–50 μg/ml. The extraction recovery from serum spiked with meropenem was 99.7±3.4%. The intra- and inter-assay coefficients of variation were below 6%. Stored at −80°C for three months at various concentrations in serum and in aqueous solution, meropenem did not reveal any appreciable degradation. After 24 h, it was also stable at 4°C in serum, aqueous solution and supernatant of extraction but not at room temperature. The stability of the drug was also confirmed in serum after repeated freezing-thawing cycles at −80°C on four consecutive days. 相似文献