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. The enzymatic rates were dependent upon Triton concentrations. Upon increasing its concentration, this detergent exhibited an activating effect followed by an inhibitory one. The optimal micellar Triton concentration was proportionnal to the PI concentration used in the assay. Conversely, the behaviour of the enzyme towards PI was dependent upon the Triton concentration. However, when PI concentration was expressed as its surfacic concentration within the micelles, the activity became independent of the detergent concentration, and a Km value of 0.09 mol/mol was estimated. Therefore, in vitro phosphorylation of phosphatidylinositol by PI kinase is rate-limited by an intramicellar reaction, and provides a study model for the in vivo reaction.

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. Further characterization of the burst will allow studies of the functional interaction of receptor-adenylate cyclase components in C6 membranes.

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. The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a Km of 1.5 +/- 0.1 microM. This transport occurred by a cooperative process with a Hill coefficient close to 2 for DNR. A model is discussed in which P-gp pumps two molecules of drug per turnover.

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. Replating of the astrocytes after a few days of culture in vitro induces an up-regulation of the receptors. This effect is particularly important when astrocytes are maintained for 6 days in the presence of horse serum, a condition that favors cellular differentiation. Only 30-50% of the beta-adrenergic receptors of the intact cells can be detected on membrane preparations. When membranes are prepared from astrocytes grown either in the presence of horse serum or under chemically controlled medium (i.e. under differentiation promoting conditions) two classes of binding sites for [125I](-)-iodocyanopindolol are revealed. Several hypotheses, mainly related to the morphology of the cells, may provide an explanation for such differences. Studies of the pharmacological specificity of receptors of membrane fractions show that cerebellar astrocytes cultured in vitro exhibit both beta 1 and beta 2 receptor subtypes. The beta 1 subtype receptors are slightly more abundant when astrocytes are grown in fetal calf serum (FCS), a condition under which they exhibit a polygonal, poorly differentiated morphology. When culture conditions favor cellular differentiation, more receptors of the beta 2 subtype are seen, which can be related to what is observed in the adult in vivo where the astrocytes exhibit a differentiated morphology.

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. Efficient DNA entrapment in preformed liposomes was obtained using the freeze/thawing procedure, and the doxorubicin passive diffusion was quantified in the presence of ATP/Mg2+, the second substrate of P-glycoprotein. The doxorubicin diffusion rate decreases in the presence of ATP, indicating an interaction between doxorubicin and ATP that will hinder any measurement of ATP-driven transport. The interaction between doxorubicin and ATP was studied by fluorescence quenching, octanol/buffer partition coefficient, and diffusion rate into DNA-loaded liposomes. The results give evidence for complex interactions. However, under our experimental conditions, these interactions are only slightly modified in the presence of Mg2+. Since this cation is essential for P-glycoprotein activity, it can be concluded that in these conditions the accurate evaluation of P-glycoprotein-catalyzed doxorubicin transport will be obtained from the Mg2+-sensitive transport into DNA-loaded proteoliposomes.

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. The initial fractional rate of increase of k(cat.) as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl beta-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in k(cat.) at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pK(a) approx. 9.

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. The other form is described and characterized herein. This latter form accounts for at least 50% of the acetylcholinesterase activity of the tissue. This enzyme associated with the tissue phospholipids. It aggregates in aqueous solution but readily dissociates to dimers in 1% sodium cholate solution, a solvent in which it is both soluble and catalytically fully active. The same dimer is obtained in sodium dodecyl sulfate solution where the enzyme is denatured. Denaturation in the presence of the reductant dithiothreitol results in the formation of a single 80000-dalton subunit. The purified enzyme contains no collagenous component. It is not derivable from the collagenous "tailed-enzyme" form in the tissue homogenate. However, the two enzymes have similar molecular weight catalytic subunits and the same substrate-dependent turnover numbers (per active site) for a variety of choline esters which are generally utilized to distinguish specific esterase function. In the tissue homogenate each form of the enzyme is associated with a characteristic structural component (phospholipid or collagen). By implication, acetylcholinesterase function is localized in situ in the phospholipid membrane as well as at the basal lamina.

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. Polymyxin B (a protein kinase C inhibitor) mimics the actions of 5-HT on fluid reabsorption and on calcium-dependent cAMP accumulation. This suggests the presence of other sources of calcium and a possible relationship between several transduction systems within different rectal cells. The second messenger system mediating the 5-HT antidiuretic message differs from those involved in the transduction of the known locust antidiuretic hormones.

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 3.2.1.23) by a point mutation, were investigated. A large decrease of the specificity, as evaluated by the kcat/Km ratio, was observed, principally originated by a weaker binding of the substrates. The catalytic constants, whose values are strongly affected by the presence of divalent cations, were smaller or larger for mutant enzyme than for wild-type enzyme, depending upon the experimental conditions. Analysis of the kinetic pathway indicates, with some substrates, a change in the limiting step for the mutant enzyme compared to the wild type. Because the k'3 step is rate limiting for hydrolysis of p-nitrophenyl-beta-D-galactoside by the mutant enzyme in the absence of Mg2+ and its value is relatively small, it is possible to observe a burst of p-nitrophenol during hydrolysis. This provides conclusive evidence for the occurrence of a two-step mechanism, with a sequential release of the products.

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 other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.

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. Nunez, eds.), Vol. 4, pp. 1-24, Elsevier/North-Holland, Amsterdam/New York (1980)] allowed its use in the determination of adenylate cyclase activity, which was otherwise precluded by high blank values. These high values originate mainly from chemically formed cyclic AMP and from ATP cross-reactivity. The simultaneous presence of ATP and magnesium ions generates cyclic AMP under the alkaline conditions used to succinylate the sample; this interference can be dealt with either by chelation of Mg2+ ions with EDTA during succinylation or by periodic acid oxidation of samples prior to succinylation. In addition, ATP itself contributes to blank values by its cross-reactivity, especially when working with high concentrations of this substrate. This interference can be decreased by a batch adsorption of ATP or oxidized ATP on alumina. Detailed procedures were discussed, with the choice of the additional steps to the standard method of Cailla et al. having to be made on the basis of the sensitivity requirements. When preventing ATP cyclization, the radioimmunoassay was as sensitive as methods using [alpha-32P]ATP as substrate. Elimination of ATP can improve the sensitivity by one order of magnitude. This method is especially interesting with high ATP concentrations and/or with low cyclic AMP production.

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. Intercalation of this dye in a poly dA-dT 12-18 template-primer was studied spectrophotometrically under conditions similar to those in the in vitro DNA polymerase assay. The polymerase assay. The inhibition by this drug of the mitochondrial DNA polymerase gamma activity was shown to be competitive at varying concentrations of TTP while the inhibition was of the non-competitive type at different concentrations of poly dA-dT 12-18. We conclude that the drug, most probably in the intercalated form, is able to interact with the active site (s) of mitochondrial DNA polymerase.

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 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP-dependent regulations in higher plants.

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.

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. Astroglial uptake was quantified by measuring the radioactivity taken up by the cells in the culture and relating this measurement to the number of glial fibrillary acidic protein-positive cells present. Glutamate uptake was investigated in astroglial cultures and subcultures and in neuronal-astroglial cultures derived from postnatal day 4 mouse cerebella. In the absence of neurons, glutamate uptake increased during the first 9 days after plating and then leveled off. At 14 days in vitro in horse serum, which favors the differentiation of fibrous-like astrocytes, glutamate uptake related to astrocyte number was twice as high as in fetal calf serum. In the presence of cerebellar neurons, this rate was even higher. The specificity of the responsiveness of astrocytes to neurons with respect to glutamate uptake was investigated by comparing GABA uptake in the different culture conditions. Neurons also increased the rate of GABA uptake by astrocytes. Another component of the astroglial plasma membrane, the density of beta-adrenergic receptors, was, however, not markedly affected by the presence of neurons. Hence, these results showed that in astrocytes plated from postnatal day 4 mouse cerebella, the level of neurotransmitter uptake can be regulated in vitro by factors present in sera and by cerebellar neurons in the culture. However, this plasticity declined during development because astrocytes plated from postnatal day 8 cerebella and cultured under identical conditions were less active in glutamate uptake and were insensitive to the presence of horse serum. The latter observation suggested that the metabolic plasticity of astrocytes is restricted to a period defined early in cerebellar development and is no longer evident by postnatal day 8.