Juta K. Reed

Simultaneous isolation of total cellular RNA and DNA from tissue culture cells using phenol and lithium chloride

A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases.

The involvement of ecto-ATPase activity in the phosphorylation of intracellular proteins by the addition of extracellular [32P]ATP in PC12 cells

Abstract We have investigated the possibility that ecto-phosphorylation by extracellular ATP may play a role in the development of PC12 cells. To test this model and to identify putative target membrane proteins, intact PC12 cells were radiolabeled by the addition of 20 μM [γ- 32 P]ATP. An analysis of the labeled proteins revealed that a 57 kDa protein was the most abundant phosphorylated protein even within time periods as short as 3 min and continued to be labeled over and above the level of other proteins. This protein was identified as tyrosine hydroxylase by immunoprecipitation with antiserum to tyrosine hydroxylase. When intact cells were incubated with either [γ- 32 P]ATP or 32 P i of comparable specific radioactivity, the overall protein labeling pattern and the degree of phosphorylation of tyrosine hydroxylase were similar. There were no discrete proteins that were labeled by [γ- 32 P]ATP and not by 32 P i that would provide evidence for ecto-kinase activity in PC12 cells. Also, the addition of nonradioactive P i reduced the incorporation of radioactivity into the protein from extracellular [γ- 32 P]ATP. These results suggested that the phosphorylation of tyrosine hydroxylase by extracellular [γ- 32 P]ATP required the initial hydrolysis of ATP and the subsequent incorporation of the 32 P i into the intracellular ATP pool. To support this interpretation, we have demonstrated directly the presence of ecto-ATPase activity in intact PC12 cells by measuring the hydrolysis of extracellular [γ- 32 P]ATP. Nearly 50% of the total ATP added (20 μM) was hydrolyzed within 10 min under conditions identical to those used to demonstrate intracellular protein phosphorylation. PC12 cells express both a Ca 2+ -dependent ecto-ATPase activity and a Mg 2+ -dependent ecto-ATPase activity. In addition, extracellular ATP is degraded enzymatically not only to ADP, but sequentially to adenosine. Our results also point out the difficulties inherent in attempts to identify ecto-kinase activity in cells that also contain ecto-ATPase activities.

Interaction of substituted guanidines with the tetrodotoxin-binding component in Electrophorus electricus.

Abstract In efforts to understand the molecular properties of ion channels in biomembranes, we have investigated the interaction of substituted guanidines with the Na+ channel site in membranes isolated from Electrophorus electricus. This interaction was measured by equilibrium competitive binding studies with [ 3 H ]tetrodotoxin ([ 3 H ]TTX); TTX has been shown to bind specifically to the Na+ channel in electrically excitable membranes. Although guanidine and small substituted guanidines such as methylguanidine or aminoguanidine competed with [ 3 H ]TTX for the membrane binding site, the apparent KI values for these derivatives were nearly seven orders of magnitude higher than the Kd for TTX. On the other hand, the binding of the guanidines was considerably enhanced by introducing a substituent aromatic ring or aliphatic chain. Detailed analysis of the binding of aliphatic guanidines of varying chain length clearly demonstrated the contribution made by hydrophobic interactions. These results suggest that the channel site may include a hydrophobic region in close proximity to the carboxylate previously postulated to be involved in TTX binding.

THE FLUORESCENCE PROPERTIES OF ORTHO AMINOBENZOATE ANESTHETICS IN DEFINED SOLVENTS AND PHOSPHOLIPID VESICLES

Abstract— The fluorescence properties of three ortho aminobenzoate local anesthetics have been determined in a variety of solvents. Results from these studies have been used to deduce how these drugs interact with phosphatidylcholine bilayers. The emission energy, fluorescence quantum yield and lifetime exhibited a biphasic dependence on solvent polarity. In aprotic solvents, alcohols and in ethanol‐water mixtures containing less than 40% water, quantum yields and lifetimes were high (approximately 0.55 and 8.5 ns respectively). In ethanol‐water mixtures containing >40% water, the strong fluorescence quenching was primarily due to an increase in the rate of non‐radiative deactivation of the excited state. Both the radiative (kr) and non‐radiative (knr) rate constants show a biphasic dependence on solvent polarity. These studies suggest the presence of two singlet excited states for these molecules, a polar singlet excited state, S1‐p and a charge transfer excited state, S1‐ct with the latter predominating in ethanol‐water mixture containing >40% water. In egg phosphatidylcholine bilayers, the fluorescence, lifetime and quantum yield are consistent with the view that these drugs are localized within the lipid head group region where the charge‐transfer excited state can be stabilized by intermolecular hydrogen bonding.

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A2 treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [3H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A2 from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by beta-bungarotoxin. (2) Phospholipase A2 from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A2 treatment of [3H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.

Interaction of a fluorescent procaine analogue with phosphatidylcholine vesicles.

Although the effects of amine local anesthetics on electrical impulse transmission are well documented, the nature of the molecular interactions between these compounds and the cell membrane is still unclear [ 1,2]. Association of local anesthetics with lipid bilayers is believed to play at least some role particularly since many physical properties such as phase behavior and fluidity appear to be sensitive to anesthetics [3,4]. On the other hand specific interactions with membrane receptors involved in excitation have also been suggested [5]. Techniques such as NMR, ESR and fluorescence spectroscopy have been used to study the mechanism and site of interaction of local anesthetics with artificial membranes [6-81. With the exception of recent NMRstudies on deuterated procaine [9], those methods often rely on the perturbation of the environment by the anesthetic as detected by incorporated spin or fluorescent probes. Although such well-studied local anesthetics as procaine and tetracaine are weakly fluorescent, their spectral properties are not suitable to allow their use directly as fluorescent probes particularly in natural membranes where spectral overlap between these molecules and protein tryptophans makes unambiguous interpretation exceedingly difficult. Nevertheless, it would be of great value to be

Characterization of the lipid and polypeptide components of a tetrodotoxin binding membrane fraction fromElectrophorus electricus

SummaryThis paper reports an analysis of the lipid and polypeptide composition of a tetrodotoxin (TTX)-binding plasma membrane fraction of the eel electroplaque. Phospholipids comprise 73% of the total lipid with cholesterol and neutral glycerides constituting about 21 and 6%, respectively. The major phospholipids are phosphatidylcholine (47.3%), phosphatidylethanolamine (32.6%), phosphatidylserine (13.1%), and sphingomyelin (4.5%). Phosphatidylinositol and phosphatidic acid are minor components. Plasmalogens comprise approximately 19% of the total phosphatidylethanolamine. Each major phospholipid class was analyzed for fatty acyl composition. The results indicate a unique distribution profile for each class with respect to chain length and unsaturation. PE and PS both contain high percentages of polyunsaturated fatty acids particularly docosahexaenoic acid with constitutes 35 and 39% of the total fatty acids, respectively. However, PC and PS contain significantly lower levels of polyunsaturated fatty acids. The lipid profile observed in this preparation is compared to those previously reported for membranes from other excitable tissues. Polyacrylamide gel electrophoresis of the membranes indicates a complex distribution of peptides with several major species and at least 30 minor components. Two of the major species have molecular weights corresponding to those of the two subunits of the (Na++K+)-ATPase.

The Synthesis of Cyclonucleotides with Fixed Glycosidic Bond Linkages as Putative Agonists for P2-Purinergic Receptors

Abstract Cyclonucleotides with fixed glycosidic bond linkages were investigated as possible ligands for purinoceptors in PC12 cells. P2Y2-purinoceptors were not activated by the ATP analogue, 8,2′-thioanhydroadenosine-5′-triphosphate (4) and only weakly by the UTP analogue, 2,2′-anhydrouridine-5′-triphosphate (6). However, both analogues were agonists for P2X2-purinoceptors although the potencies were approximately 30-fold less than that of the parent nucleotides.