Partition Assay Research Articles

Isolation and partial characterization of the surface protein of Lactobacillus acidophilus strains

A surface protein layer was found to be present in some Lactobacillus acidophilus strains. it could be visualized with electron microscopy and extracted with sodium dodecyl sulfate or guanidine hydrochloride. The protein was resistant to several proteolytic enzymes, but sensitive to pronase. Amino acid composition analysis revealed about 30% hydrophobic amino acids. Hydrophobicity study with a hexadecane partition assay demonstrated that the cells were hydrophobic and removal of the surface layer with pronase destroyed the hydrophobic nature of the cells. Several growth conditions studies were found to have little effect on synthesis of the surface protein.

Improved version of the Kean partition assay for cerebroside sulfate.

In the Kean method for the colorimetric determination of sulfatide (1968. J. Lipid Res. 9: 319-327), the lipid is partitioned together with a blue cationic compound between two phases formed from chloroform, methanol, and water. The blue cation enters the chloroform-rich phase only as an ion pair with the lipid. This method has been improved by the use of a new mixture of solvents in which the desired layer floats above the excess dye. The lower volatility of the new solvent system improves the reproducibility of the technique.

Juvenile hormone metabolism, binding and esterase activities in the haemolymph of the adult monarch butterfly ( Danaus p. plexippus L.: Lepidoptera)

The interaction of [ 3H]juvenile hormone I (JHI) with haemolymph of the adult monarch butterfly, Danaus p. plexippus L. was investigated. Whole haemolymph bound [ 3H]JHI preferentially in vitro compared to bovine serum albumin or buffer alone using a charcoal binding assay. [ 3H]JHI was converted to polar metabolites by adult haemolymph as assessed by an octane: methanol-H 2O partition assay and thin layer chromatography. Phenyl methyl sulphonyl fluoride (PMSF), a potent esterase inhibitor, was effective in decreasing the conversion of [ 3H]JHI to polar metabolites, thus suggesting the presence of JH esterase (JHE) activity. Post eclosion JHE activity assessed in vitro increased in females, peaking at day 6 post eclosion; while in males there was an initial decrease followed by a small peak at day 6. The fluctuations observed were not attributable to haemolymph volume changes. [ 3H]JHI metabolism assessed in vivo at 40, 140, 240 and 1380 min post injection, tested at four different stages or periods of the life cycle, indicated that the greatest metabolism of [ 3H]JHI occurred at day 3 post eclosion in both sexes followed respectively by January males and females, June-July males and females, September females and finally September male wild caught adults. JH binding assays carried out on haemolymph fractions separated by Sephadex G-100 column chromatography indicated a peak of JH binding activity corresponding to 29,500 ± 6000 mol. wt ( x ± SEM , n = 6). Bioassay indicated that the bulk of endogenous JH was associated with this peak of JH binding activity. JHE activity assessed in these same fractions indicated two major peaks one > 100,000 mol. wt and the other at 79,000 ± 5000 mol. wt ( x ± SEM , n = 6). The fractions of the smaller mol. wt peaks were found to inactivate both [ 3H]JHI and endogenous JH biological activity when assessed using the Galleria wax test.

Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid:Coenzyme a ligase activities in rat liver.

Cholic acid:CoA ligase (EC 6.2.1.7, choloyl-CoA synthetase) and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were found to be localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent maximal velocities for the two substrates are similar, the Michaelis constant for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are shown to be competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Michaelis constant for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.

Induction of hepatic gamma-glutamyl transpeptidase in rats by repeated administration of aminopyrine

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman′s method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen β to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.

A rapid partition assay for routine analysis of juvenile hormone release by insect corpora allata

A partition assay is described which allows a rapid determination of juvenile hormone release by insect corpora allata. Newly synthesized and released methyl-labeled C 16 juvenile hormone is quantitatively extracted into an isooctane phase, whereas precursor methyl-labeled l-methionine remains in the incubation medium. When used in conjunction with appropriate blank assays, this method offers both accuracy and speed necessary for routine analysis of corpora allata activity.

Partition of hexadecane to the cell surface of Candida tropicalis: Mechanism for the transport of water‐insoluble substrates

AbstractThe partition of hexadecane to the cell surface of Candida tropicalis was measured by incubating heat‐inactivated cells with hexadecane‐1‐14C on a gyratory shaker. The free hexadecane was separated by centrifuging the cells through a 15% sucrose solution, and the partitioned hexadecane was quantified by scintillation spectrometry of the samples from the resulting cell sediment. Heat‐inactivated cells did not take up hexadecane as determined by a membrane filtration technique involving organic solvent washing. The partitioning was a time‐dependent process. The velocity increased by increasing the shake rate of te shaker. At 360 rpm and with baffled flasks, saturation of the cell surface with hexadecane was obtained after a 20 min incubation period. The amount of hexadecane partitioned depended on the initial hexadecane‐to‐cell concentration ratio. At a ratio of 5 μmol/mg cell protein the highest amount of hexadecane partitioned was measured at 2100 μmol/mg cell protein. At ratios higher than 6 μmol/mg cell protein the cells were no longer sedimentable by centrifugation. The partition of hexadecane to the cell surface was affected by removing the surface layer of the cell wall by Pronase treatment and by using detergents in the partition assay. Pronase treatment lowered the amount of hexadecane partitioned as a consequence of the removal of the lipophilic layer of the cell surface. Detergents influence the partition coefficient and also lowered the amount of hexadecane partitioning to the cell surface. At a low shaking intensity (280 rpm, unbaffled flasks), after Pronase treatment, and in the presence of detergents he uptake of hexadecane by the cells was limited by the partitioning.

A partition assay for epoxide hydrases acting on insect juvenile hormone and an epoxide-containing juvenoid

An assay for epoxide hydrase activity is described based on the difference in solubility between the epoxide substrate and its corresponding diol. The assay conditions for insect juvenile hormone I and an epoxide-containing juvenoid have been elucidated. In each case, the epoxide partitions into an isooctane phase while the diol partitions largely into an aqueous methanol phase. The assay is faster than chromatographic assays while showing similar or superior precision.

Determination of Procaine and Related Local Anesthetics. I. Partition Chromatographic Separation and Assay of Mixtures of Procaine with Tetracaine and with Propoxycaine

Determination of ionization and extraction constants for procaine, tetracaine, and propoxycaine led to selection of a simple partition chromatographic system for separation and assay of mixtures of these anesthetics. A 65% solution of chloroform in isooctane elutes tetracaine or propoxycaine from a pH 4:sodium bromide column; procaine is retained and subsequently eluted by chloroform as the bromide ion-pair. The anesthetics are then determined spectrophotometrically. Results of assay of standard and commercial formulations are presented.