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PubMed1999

High-performance liquid-phase separation of glycosides. III. Determination of total glucosinolates in cabbage and rapeseed by capillary electrophoresis via the enzymatically released glucose.

A selective and sensitive method for the determination of total glucosinolates (GSs) in plant extracts by capillary electrophoresis-laser-induced fluorescence (LIF) detection was developed. It was based on the enzymatically released glucose from glucosinolates in the presence of the hydrolyzing enzyme myrosinase. The released glucose was converted to gluconic acid (GA) by the action of glucose oxidase. The resulting GA was then labeled selectively with the fluorescent tag 7-aminonaphthalene-1, 3-disulfonic acid (ANDSA). The peak area resulting from the GA-ANDSA derived from free and bound glucose was subtracted from the peak area of the GA-ANDSA resulting from the free glucose in the sample. This gave the total glucosinolates in the sample. The peak areas were normalized to the internal standard, N-acetylneuraminic acid derivatized with ANDSA. The method was validated using four different plant extracts, white cabbage leaves, rapeseed leaves, rapeseed roots, and rapeseed seeds. Furthermore, a capillary electrophoresis-UV detection method for profiling GS in plant extracts was developed. In addition to providing a fingerprint of the glucosinolates in plant extracts, the method allowed the experimenter to rapidly check the various steps involved in the extraction and sample cleanup.

2-Naphthylamine↗
PubMed1989

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes.

The association of [125I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25 degrees in the presence or in absence of 20 microM Ca2+, follows a sigmoid path with a Hill coefficient h = 1.79 +/- 0.12 and h = 1.72 +/- 0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%-80% at all the range of calmodulin concentrations used in the presence of 20 microM Ca2+. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 nM); higher concentrations (up to 40 nM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the membrane at 22.7 +/- 1.2 degrees was perturbed by calmodulin such that the temperature was reduced to 16.3 +/- 0.9 degrees and 15.5 +/- 0.8 degrees in the absence or in the presence of 20 microM Ca2+. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited break points at 26.7 +/- 1.4 degrees and 22.3 +/- 1.0 degrees in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3 +/- 0.9 degrees in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1 +/- 0.9 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1984

Lateral phase separation of phospholipids as a basis for increased permeability of membranes towards fluorescein and other chemical species.

Using mouse spleen cells, before and after treatment with glutaraldehyde or mild hyperthermia, we observe a strong correlation between permeability to fluorescein and susceptibility to staining with N epsilon-dansyl-L-lysine (irrespective of the cells' ability to exclude trypan blue). We observe the same correlation using liposomes prepared from phosphatidylcholine and varying amounts of cholesterol. We have recently introduced N epsilon-dansyl-L-lysine as a fluorescent membrane stain, or "probe," whose uptake, we propose, is selective for phospholipid domains in membranes (G.M.K. Humphries & J.P. Lovejoy Biophys. J. 42:307-310, 1983; G.M.K. Humphries & J.P. Lovejoy J. Membrane Biol. 77:115-122, 1984). The results presented here are consistent with the hypothesis that the presence or absence of phospholipid domains in membranes also modifies their permeability toward fluorescein, and suggests that permeability towards other chemical species may be similarly affected. On the basis of work using liposomes, we believe this to be the case for carboxyfluorescein and for glucose.

Animals↗
PubMed1987

A novel immunoassay system and bioseparation process based on thermal phase separating polymers.

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyNIPAAm precipitates out of water above a critical temperature of 31 degres C, enabling a polymer-bound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.

Acrylic Resins↗
PubMed2004

Enantiomer discrimination by mass spectrometry: noncovalent interactions of an N-derivatized dipeptide with various cinchona alkaloid derivatives and comparison with enantioselective liquid-phase separations.

The enantiomer discrimination properties of cinchona alkaloid derived chiral selectors (CSs) towards a dipeptide analyte are examined by electrospray ionization mass spectrometry. The complexes formed between the CSs and the analyte enantiomers owing to various noncovalent interactions are analyzed and the magnitudes of enantiomer discrimination are determined from the complexes' mass spectrometric intensities. The influence of different structural features of the CSs on enantioselectivity is discussed. The enantiomer discrimination results obtained by mass spectrometry are compared with those from related liquid chromatography enantiomer separations. A certain coherence between the chromatographic and mass spectrometric enantioselectivities could be established and the enantiomer discrimination patterns, i.e., the relative binding strengths, were identical for the two techniques. Thus, the use of mass spectrometry as a screening tool in the development of new CSs for chromatographic applications seems feasible.

Chromatography, Liquid↗
PubMed2001

Gas-phase separations of complex tryptic peptide mixtures.

High-resolution ion mobility and time-of-flight mass spectrometry techniques have been used to analyze complex mixtures of peptides generated from tryptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pig, and sheep; aldolase, rabbit; beta-casein, bovine; cytochrome c, horse; beta-lactoglobulin, bovine; myoglobin, horse; hemoglobin, human, pig, rabbit, and sheep). In this approach, ions are separated based on differences in mobilities in helium in a drift tube and on differences in their mass-to-charge ratios in a mass spectrometer. From data recorded for fourteen individual proteins (over a m/z range of 405 to 1,000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digestion. In a separate analysis, the fourteen mixtures have been combined and analyzed as one system. In the single dataset, we resolve 260 features and are able to assign 168 peaks to unique peptide sequences. Many other unresolved features are observed. Methods for assigning peptides based on the use of m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described.

Amino Acid Sequence↗
PubMed1980

Pressure-anesthetic antagonism on the phase separation of non-ionic surfactant micelles.

An aqueous solution of non-ionic surfactants becomes suddenly turbid when heated to a critical temperature, known as the cloud point, and concomitantly expands the volume. The volume expansion is caused by release of structured water molecules from the hydrophilic polyoxyethyelene moieties. Inhalation anesthetics decreased the cloud-point temperature of hexaoxyethylene dodecyl ether micelles. The concentrations of methoxyflurane, halothane and enflurane causing a 1 degree C depression of the cloud-point temperature were 0.51, 0.71 and 0.78 mmolal, respectively. Hydrostatic pressure increased the cloud-point temperature in the absence and presence of the anesthetics. The change of the apparent molal volume at the cloud point was estimated to be 2.2 cm3/mol in the absence of anesthetics. This value decreased in the presence of the anesthetics, dose dependently. The results indicate that the anesthetics favor dehydration of the hydrophilic surface of the non-ionic surfactant micelles.

Anesthetics↗
PubMed1981

Fluidity of human erythrocyte membrane and effect of chlorpromazine on fluidity and phase separation of membrane.

The fluidity of human erythrocyte membrane, and the effect of chlorpromazine at prelytic and lytic concentrations on the fluidity have been studied by using three kinds of fatty acid spin labels and measuring the temperature dependence of Mg2+-ATPase activity. The Arrhenius plot of the apparent rotational correlation time, tau c, for probes I(12,3) and I(5,10) showed an abrupt discontinuity at about 30 degrees C, and the plot for I(1,14) at 25 degrees C, indicating that a large difference in the fluidity exists between the interior and the outer surface of the lipid bilayer. The portions of the fatty acid chain near the ten carbon bond lengths removed from the bilayer surface became more fluid by chlorpromazine treatment; there was a decrease in the break point to around 26 degrees C following treatment with 0.6 or 1 mM of the drug. Two breaks at 21 and 30 degrees C in the Arrhenius plot of the Mg2+-ATPase activity were observed in normal erythrocyte membrane. The activation energy of the Mg2+-ATPase reaction has the values of 3.0 and 22.1 kcal/mol above the upper break and below the lower break, respectively. The drug exposure induced only a slight shift in the break temperatures, while the treatment significantly enhanced the associated activation energies of the reaction. These results suggest that the boundary phospholipids of the Mg2+-ATPase in the membrane are probably more rigid than the bulk lipids.

Adenosine Triphosphatases↗