Polypyrrole-glucose oxidase biosensor. Effect of enzyme encapsulation in multilamellar vesicles on analytical properties.

In this paper, we present the analytical properties of a new type of polypyrrole-based, enzymatic amperometric biosensor. It is produced by encapsulating the enzyme, glucose oxidase (GOx), into onion-type multilamellar vesicles (MLV). We compare its properties to those of a classical GOx-polypyrrole biosensor.

Interaction of doxorubicin with ATP: quantification of complexes and effect on its diffusion into DNA-loaded liposomes--implication for ATP-driven transport studies.

Doxorubicin, a drug largely used in chemotherapy, is transported by P-glycoprotein, a protein involved in the multidrug-resistance phenotype. Taking advantage of the doxorubicin fluorescence quenching upon interaction with DNA, a sensitive assay of this active transport can be carried out: quantitative in vitro studies could be achieved with DNA-loaded proteoliposomes, after correction for the doxorubicin passive diffusion through phospholipids. In this paper, we describe experimental conditions that will be relevant to P-glycoprotein studies.

Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles.

Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump. Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport. This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles.

Plasticity of astroglial glutamate and gamma-aminobutyric acid uptake in cell cultures derived from postnatal mouse cerebellum.

The plasticity of astroglial glutamate and gamma-aminobutyric acid (GABA) uptakes was investigated using mouse cerebellar cell cultures. The influence of external factors, such as different sera and/or the presence of neurons, was examined. Control autoradiography experiments showed that after short-term exposure to radioactive amino acids, granule cells took up neither glutamate nor GABA, and beta-alanine predominantly inhibited astroglial GABA uptake.

Neurotransmitters and stimulation of fluid reabsorption in migratory locust rectal cells.

Several biogenic amines enhance fluid reabsorption and the accumulation of cyclic adenosine-monophosphate (cAMP) in the rectum of the migratory locust but only 5-hydroxytryptamine (5-HT) acts in a dose-dependent manner at low concentrations (between 10(-8) and 5.10(-7) M). Cyclic AMP is a second messenger of 5-HT, and its actions on fluid reabsorption are calcium-dependent.

Platelet membrane phosphatidylinositol kinase activity. Triton X-100 effects provide evidence for intramicellar reaction.

Phosphatidylinositol (PI) kinase activity of platelet membranes was solubilized and partially purified by anion-exchange chromatography to measure the initial enzymatic rates. Kinetic studies were performed in the presence of Triton X-100 to obtain mixed micelles. The partially purified enzyme exhibited a Michaelian behaviour towards ATP, with a Km of 58 microM.

Artefactual Origins of Cyclic AMP in Higher Plant Tissues.

A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3',5'-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the characterization of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.

A quenched-flow study of a receptor-triggered second messenger response: cyclic AMP burst elicited by isoproterenol in C6 glioma cell membranes.

Fast kinetic studies of cAMP accumulation in C6 cell membranes show a burst of cAMP after beta-adrenergic receptor stimulation by isoproterenol. This burst is no longer observed when the ATP present in membrane preparations is hydrolyzed, but can be restored by their preincubation in the presence of ATP-Mg. The size of the burst is much larger than the number of beta-adrenergic receptors and is of the same order of magnitude as the value reported for G proteins.

Beta-adrenergic receptors of cerebellar astrocytes in culture: intact cells versus membrane preparation.

Experiments were carried out to assess: the influence of culture conditions on the expression of beta-adrenergic receptors in intact glial cells from the central nervous system; and the extent to which quantitation of receptor sites in membrane preparations reflects the receptor population of the whole cells they are derived from. Cerebellar astrocytes were chosen for this study since essentially one receptor subtype, the beta 2 one, is present in adult cerebellum. Intact, attached cerebellar astrocytes exhibit only one class of binding sites for the beta-adrenergic antagonist, [3H]CGP 12177.

Radioimmunoassay of cyclic AMP can provide a highly sensitive assay for adenylate cyclase, even at very high ATP concentrations.

Modifications of the cyclic AMP radioimmunoassay of Cailla et al. [in Hormones and Cell Regulation (J. Dumont and J.

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated.

Electroplax tissue from Torpedo californica contains two major structural forms of the enzyme acetylcholinesterase. One form, composed of tetrameric protomers which are further aggregated by interactions among associated collagenous "tail fibers", has been well characterized previously. This form is associated in situ with the basal lamina.

Dependence upon pH of steady-state parameters for the beta-galactosidase-catalysed hydrolyses of beta-D-galactopyranosyl derivatives of different chemical types.

The effect of pH upon the beta-galactosidase-catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a 'non-bottleneck' pathway. While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non-covalent interactions.

The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.

DNA polymerase gamma from purified nuclei of EMT-6 cells (mice) seems to be identical to the mitochondrial DNA polymerase from the same source following several criteria. These two enzyme activities are strongly inhibited by ethidium bromide and acriflavin, while proflavin, acridine orange, daunomycin and chloroquine inhibition is less pronounced. In the case of DNA polymerases alpha and beta very little inhibition by ethidium bromide was observed.

Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.

The functional properties of CZP protein, a mutant deriving from wild-type beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition.

The effect of methanol and dioxan on the rates of the beta-galactosidase-catalysed hydrolyses of some beta-D-galactrophyranosides: rate-limiting degalactosylation. The ph-dependence of galactosylation and degalactosylation.

1. The effect of methanol on the beta-galactosidase-catalysed hydrolysis of some nitrophenyl beta-d-galactopyranosides has been studied under steady-state conditions. 2.