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At least 649 records · Page 36Linked to original sources
PubMed2004

Dialysis-Chelex method for determination of exchangeable copper in human plasma.

A method based on dialysis in the presence of histidine, and subsequent copper adsorption and preconcentration using Chelex-100 resin, has been developed for selective extraction of exchangeable copper from blood plasma. The method was verified by comparative analysis using two different instrumental techniques, ICP-MS and AAS. The results obtained were in excellent agreement. Accurate quantification of the exchangeable copper and the firmly bound copper was achieved. The method was further validated by analysis of a lyophilised human serum certified reference material.

Copper↗
PubMed2004

Membrane cholesterol regulates smooth muscle phasic contraction.

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts-and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K(+)-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its alpha-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertension.

Animals↗
PubMed2006

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production.

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas-liquid volumetric mass transfer coefficient k (L) a positively. The k (L) a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k (L) a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r (DHA) (9.75 mg l(-1 )h(-1)) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.

Alkanes↗
PubMed2005

Geochemical distribution and bioavailability of heavy metals and oxalate in street sediments from Rio de Janeiro, Brazil: a preliminary investigation.

As urbanisation accelerates within less-economically developed countries, populations in cities such as Rio de Janeiro are subject to numerous health risks relating to "heavy metal", sewage and vehicle pollution. These risks apply especially to children, through inhalation and dermal contact with pollutant-rich street sediments that reflect contamination from atmospheric deposition and act as effective sinks for heavy metals and oxalates. To assess the nature and extent of these risks street sediments were collected from industrial, commercial, residential and recreational areas with varying traffic densities within Rio de Janeiro. A modified selective extraction procedure was used to study the geochemical partitioning and bioavailability of Fe, Mn, Zn, Cu, Cr, Ni, Pb and C(2)O(4) (-). Oxalate partitioning has not been studied by traditional sequential methods and results from this procedure highlight the potential bioavailability of both oxalates and "heavy metals", especially Pb and C(2)O(4) (-) in industrial and recreational areas.

Biological Availability↗
PubMed2006

Fractionation of aluminum in soil and relation to its concentration in fruits.

Aluminum concentrations in the fruit samples taken from different regions were determined by atomic absorption spectrometry after dry ashing digestion. To identify the aluminum phases being most responsible for fruit-available aluminum, the soil samples near the fruit plants were also analyzed for aluminum by using various digestion and selective extraction reagents. The relation between the aluminum concentrations in fruits and in soil extracts was studied. The obtained aluminum concentrations in the fruits were in the range of 1.5 to 42.0 mg kg(-1) on dry weight basis. It was observed that the aluminum concentrations of morello cherry(R(2) = 0.79) and mulberry (R(2) = 0.99) were correlated to the aluminum concentrations in citric acid extracts of the soils. While the aluminum concentrations of the EDTA and acetic acid extracts in some soils samples include 35% and 25% of total aluminum, respectively, the other soils contain only 1-2%.

Aluminum↗
PubMed1985

Analysis of oxidized and reduced pyridine dinucleotides in rat liver by high-performance liquid chromatography.

NADP+, NAD+, NADPH, and NADH were assayed by selective extraction and isocratic reversephase HPLC. Sample preparation involves freeze clamping and powdering liver under liquid nitrogen, extraction of dinucleotides with basic (reduced species) or acidic (oxidized species) cold ethanol, and injection onto the HPLC for quantitation at 340 nm (reduced) and 254 nm (oxidized). The mobile phase for the oxidized species is pH 5.25, 0.2 M ammonium phosphate/methanol, and for the reduced species is pH 6.0, 0.2 M ammonium phosphate/methanol/tributylamine. The method is linear over the range 0.016 to 2.0 nmol for the reduced species, and from 0.005 to 0.8 nmol for the oxidized pyridine dinucleotides. The recoveries were from 94.5% for NAD+ to 99.3% for NADPH, with standard deviations of approximately 2.5% for all species other than NADP+, which had a standard deviation of 10.4%. The coefficients of variation for repeated determinations of standards over 3 months were less than 4%.

Animals↗
PubMed1985

Measurement of urinary 2,3-dinor-thromboxane B2 and thromboxane B2 using bonded-phase phenylboronic acid columns and capillary gas chromatography--negative-ion chemical ionization mass spectrometry.

The use of bonded-phase phenylboronic acid columns to selectively extract 2,3-dinor-thromboxane B2 and thromboxane B2 from urine is reported. The compounds were first derivatized as the methoxime and then applied to the phenylboronic acid columns. Subsequent purification by thin-layer chromatography and derivatization to the pentafluorobenzyl ester, trimethylsilyl ether followed by capillary gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring specific ions, allows quantitation in the low-picogram/milliliter range. In healthy male volunteers, the median excretions of 2,3-dinor-thromboxane B2 and thromboxane B2 were 10.3 ng/h (range, 4.5-24 ng/h) and 2.8 ng/h (range, 0.5-7.3 ng/h), respectively. The method offers a noninvasive, specific approach to the study of thromboxane synthesis and platelet function in man. It is much less labor intensive than currently available methods employing electron-impact chromatography-mass spectrometry.

Boronic Acids↗
PubMed1987

Colorimetric assay for methanesulfinic acid in biological samples.

We describe a simple colorimetric method to measure 30 to 300 microM concentrations of sulfinic acids in biologic samples. The procedure employs the coupling reaction of an aromatic diazonium salt (Ar--N = N+) with the sulfinic acids (RSOOH) to produce a colored diazosulfone derivative (Ar-N = N-SOOR), which can be selectively extracted into an organic solvent. Linearity as well as noninterference by liver homogenate, phenols, amines, and thousandfold or greater excesses of sulfate, thiol, and dimethyl sulfoxide is demonstrated. Sensitivity of the method is about 10 nmol per sample. Because methanesulfinic acid is the principal product of the action of hydroxyl radicals upon dimethyl sulfoxide, and because intact animals can tolerate dimethyl sulfoxide in millimolar concentrations, the method may prove widely useful for detecting the involvement of hydroxyl radicals in pathologic processes in vivo.

Animals↗
PubMed1987

Additives for immobilized pH gradient two-dimensional separation of particulate material: comparison between commercial and new synthetic detergents.

We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commercial compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3-12, the concentration of urea compatible with 2% detergent increases from 4.5 M for the parent molecule up to 7 M. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl-N,N-dimethylammonio-3-propanesulfonate of a major protein (pI = ca. 5.5, Mr = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a pK of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.

Animals↗
PubMed1984

Purification and properties of gentamicin nucleotidyltransferase from Escherichia coli: nucleotide specificity, pH optimum, and the separation of two electrophoretic variants.

Gentamicin nucleotidyltransferase, AAD 2", catalyzes the transfer of a nucleotide to many aminoglycoside antibiotics, which are the drugs of choice in the treatment of gram-negative bacterial infections. The transfer is accompanied by the production of pyrophosphate, which is coupled to three other enzymes so that an increase in absorbance at 340 nm of NADPH can be monitored continuously as a quantitative assay of activity. A purification method was developed for this enzyme using all common principles of protein purification. These include selection of a desirable source of enzyme (choice of plasmid pMY 10), maximizing cellular yield of enzyme (controlled and monitored growth of Escherichia coli pMY 10/W677), selective extraction of protein (modified osmotic shock), removal of nucleic acids (precipitation with streptomycin sulfate), concentration of protein (precipitation with ammonium sulfate), removal of low-molecular-weight impurities (chromatography on Bio-Gel P-2), separation of proteins on the basis of charge (ion-exchange chromatography on DEAE-Bio-Gel A), separation of proteins according to a biospecific property (affinity chromatography on gentamicin-Affi-Gel), and separation of proteins according to size (gel filtration on Ultrogel AcA 54). Purification to near-homogeneity revealed the presence of two related forms of enzyme. The first had a specific activity of 0.134 units/mg, bound rapidly and tightly to gentamicin-Affi-Gel, eluted as a function of ionic strength from Ultrogel, and migrated faster during electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 5.7 +/- 0.2 and consists of a single polypeptide of 32,500 Da. Kinetic characterization showed a pH optimum of 9.5 and Michaelis constants of 2.76 +/- 0.35 microM for tobramycin, 404 +/- 28 microM for Mg-ATP, 2008 +/- 260 microM for Mg-CTP, 30 +/- 3 microM for Mg-dATP and Mg-dGTP, and 90 +/- 7 microM for Mg-dCTP and Mg-dTTP. The second form had a specific activity of 0.274 unit/mg. It also bound tightly to gentamicin-Affi-gel but the onset of binding was time dependent. This form migrated slower during polyacrylamide gel electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 6.0 +/- 0.2 and consists of a single polypeptide of 31,500 Da. The exact relationship between the two forms has not been elucidated. It is probable that they have a recent common ancestor or are the same polypeptide because the amino acid compositions and polypeptide chain lengths are essentially identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Phenomena↗
PubMed1994

Topological studies of spinach 22 kDa protein of Photosystem II.

An intrinsic 22 kDa polypeptide is associated with the O2-evolving Photosystem II core complex in a variety of green plants, although it does not appear to be required for O2 evolution. Digestion of thylakoid membranes and isolated Photosystem II preparations with trypsin, followed by immunoblotting using spinach anti-22 kDa antibodies, leads to two observations: (1) the domain between the 2nd and 3rd transmembrane helices of the 22 kDa protein is stromally exposed, and (2) only in a reaction center complex preparation, lacking the chlorophyll a/b-light harvesting complex II, is there extensive proteolytic cleavage of the 22 kDa protein. We also found that after, but not prior to, selective extraction of the 22 and 10 kDa proteins from Photosystem II membranes, the chlorophyll a/b-light harvesting complex II can be separated from the Photosystem II reaction center core by precipitation with MgCl2. This result suggests that the 22 kDa polypeptide is located between the Photosystem II reaction center polypeptides and light-harvesting complex II; it is possible that the protein serves as a link between the two protein complexes. The presence of the 22 kDa protein in several species was also examined by immunoblotting with polyclonal spinach anti-22 kDa antibodies.

Amino Acid Sequence↗
PubMed1975

Non-parallel transport of membrane proteins and content proteins during assembly of the secretory granule in rat parotid gland.

The insertion of newly synthesized protein molecules into the membrane of the secretory granule of the rat parotid gland was studied by in vivo labeling with [3-H]-proline and [3-H]leucine. 2 h after the injection of the amino acid into the rat, the membrane fraction isolated from the secretory granules was found to be highly labeled with proline but only slightly labeled with leucine. The ratio of proline label in the granule membrane to that in the granule's secretory content was roughly equivalent to the ratio of total proline in the proteins of these two fractions. In contrast the ratio of leucine label in the membrane to that in the secretory content was much less than would be expected from the relative amount of leucine in both fractions. Separation of the proteins of the granule membrane by gel electrophoresis in presence of sodium dodecylsulfate showed that a considerable amount of these proteins was unlabeled. The labeled proteins could be selectively extracted from the membrane by 0.15 M Nacl solution or by dilute buffer at pH 4.5. These extracted proteins were found to contain a high proportion of proline residues and a negligible amount of leucine residues. In the extract proline constituted 36 mole % of the total amino acids. Proline plus glycine plus glutamic acid constituted more than 80 mole % and leucine constituted about 1 mole% of the total amino acids. Further analyses by gel electrophoresis in presence of sodium dodecylsulfate showed that the fractions of secretory granule membrane and secretory granule content are relatively free of contamination by proteins from other subcellular structures. It is suggested that the proteins which will constitute the mature secretory granule are transported to the site of final assembly by two pathways. The proline-rich proteins are transported to the site of assembly in close coordination with all the exportable proteins. The other membrane proteins arrive by a different pathway. Two alternative mechanisms are suggested to explain the finding that a considerable part of the membrane proteins are not labeled. I. The pathway of the intracellular transport of the unlabeled membrane proteins is similar to that of the secretory proteins but the newly synthesized membrane protein molecules are diluted in a large intermediate pool--the GOLgi complex. II. The proteins that did not get labeled are derived by a process of reutilization, from membranes of granules which have previously discharged their content in the process of secretion.

Amino Acids↗
PubMed1975

Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine.

The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.

Adenosine Triphosphatases↗
PubMed1980

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent ('basal') ATPase activity.

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca2+-ATPase is selectively extracted while approximately half of the initial Mg2+-, or 'basal', ATPase remain in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca2+-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg2+-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the 'basal' ATPase separated from the Ca2+-ATPase by detergent extraction originates from mitochondrial contaminants. To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90-95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg2+-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca2+-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.

Adenosine Triphosphatases↗
PubMed1980

Two-dimensional gel electrophoresis of rat liver microsomal membrane proteins.

Total rat liver microsomal proteins are not suitable for isoelectric focusing in polyacrylamide gels, even in the presence of sodium dodecyl sulphate and excess non-ionic detergent; considerable quantities of protein form an aggregate in the isoelectric focusing gel. This prevents resolution of microsomal proteins by the increasingly popular two-dimensional electrophoresis technique employing isoelectric focusing followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The problem is caused by the extreme insolubility of some microsomal proteins, especially cytochrome P-450 species, which precipitate during isoelectric focusing. A selective extraction of microsomes with sodium deoxycholate excludes these poorly soluble proteins. The extracted proteins can then be resolved without difficulty by isoelectric focusing, and give excellent two-dimensional gel patterns showing more than 100 proteins, mainly in the pI range 5--7. The technique should be useful in studies on microsome protein topology and on changes in microsome composition.

Animals↗
PubMed1992

ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1976

A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate.

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.

Animals↗