Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Extraction of DNA from milligram amounts of fresh,herbarium and mummified plant tissues

Summary We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3–200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22–118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.     

The effect of neutral and charged micelles on the acid-base dissociation of the local anesthetic tetracaine

The influence of surfactant micelles on the acid-base dissociation of the charged tertiary amino group of the local anesthetic, tetracaine, has been investigated. From measurements of tetracaine fluorescence as a function of bulk pH, apparent $\text{p}\text{K}$ values of 6.88, 7.58 and 9.92 were found in the presence of cationic, neutral and anionic micelles, respectively, in 10 mM NaCl. These values are considerably displaced with respect to the $\text{p}\text{K}$ in aqueous solution which is 8.26. Such large shifts can be attributed to the effect of the surface polarity and electrical potential on the dissociation behavior of the anesthetic bound to micelles. It can be expected that the acid-base dissociation of a local anesthetic adsorbed to nerve fibers will also be affected by the properties of the membrane surface. Thus, it is suggested that the influence of the interfacial region on the $\text{p}\text{K}$ of surface-bound molecules should not be disregarded when estimating the proportion of charged and uncharged forms of local anesthetics interacting with axonal membranes.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

Effect of 7,8-benzolfavone on the formation of benzo(alpha)pyrene-DNA-bound products in hamster embryo cells.

The hydrocarbon-deoxyribonucleoside products present in enzyme digests of DNA from hamster embryo cultures that had been treated with[3H]-benzo[alpha]pyrene (BP) were isolated by chromatography on Sephadex LH20 columns. The products isolated from cells treated with 7,8-benzoflavone (7,8-BF) for 18 h prior to the addition of [3H] BP were indistinguishable from the products isolated from untreated cultures, but the amounts of these products decreased with increasing concentrations of 7,8-BF. The amount of BP metabolized was also decreased in 7,8-BF-treated cultures. The decrease in the amounts of hydrocarbon-deoxyribonucleoside products per mg DNA was logarithmic with respect to the decrease in BP metabolism. The findings are consistent with the hypothesis that 7,8-BF inhibits both an initial and a later metabolic step involved in the conversion of BP to a reactive species that binds to cellular DNA.     

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pK_a of the uncoupler molecule.

The absorption and fluorescence excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca²⁺ strongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes.

With mitochondrial membranes an effect of Ca²⁺ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it.

From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholipid moiety of the membrane, with its anionic group pointing to the aqueous solution.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Analysis of genetic variability and relationships among Mentha L. using the limonene synthase gene, LS

The genus Mentha comprises a group of aromatic plants with worldwide distribution. Because of frequent interspecific hybridization, the genetic relationships within the genus are not clearly understood. Limonene synthase, which catalyses the first committed step in the essential oil monoterpene biosynthetic pathway, is considered to be a possible rate limiting enzyme. With the homology-based cloning method, primers were designed according to cDNA sequence to amplify full-length DNA sequences in 13 Mentha samples from five species, using Perilla as an outgroup. Analyses of gene structure, length variation, GC-content, Ts/Tv ratio and evolutionary diversity were carried out. Consensus phylogenetic trees were obtained using maximum likelihood, neighbor-joining, and maximum parsimony, respectively, based on the full-length genomic DNA sequences, complete ORF coding sequences and predicted amino acid sequences. The results presented here based on the sequence of MhLS provide the first credibly supported genetic relationships for Mentha, which enables a basis for further mint taxonomy, cultivation and breeding.