Synthesis of phosphatidylcholine under possible primitive Earth conditions

Summary Using a primitive Earth evaporating pond model, the synthesis of phosphatidylcholine was accomplished when a reaction mixture of choline chloride and disodium phosphatidate, in the presence of cyanamide and traccs of acid, was evaporated and heated at temperatures ranging from 25° to 100°C for 7 hours. Optimum yields of about 15% were obtained at 80°C. Phosphatidylcholine was identified by chromatographic, chemical and enzymatic degradation methods. On enzymatic hydrolysis with phospholipase A₂ and phospholipase C, lysophosphatidylcholine and phosphorylcholine were formed, respectively. Alkaline hydrolysis gave glycerophosphorylcholine. The synthesis of phosphatidylcholine as the major compound was accompanied by the formation of lysophosphatidylcholine in smaller amounts. Cyanamide was found to be essential for the formation of phosphatidylcholine, and only traces of HCl, of the order of that required to convert the disodium phosphatidate to free phosphatidic acid were found necessary for the synthesis. This work suggests that phosphatidylcholine, which is an essential component of most biological membranes, could have been synthesized on the primitive Earth.     

The Effect of Anti-Synaptosomal Membrane Antibodies on Neurotransmitter Uptake

Antibodies raised against synaptosomal plasma membranes of rat hippocampus (anti-HPC IgG) caused inhibition of [3H]noradrenaline, [3H]5-hydroxytryptamine, [3H]GABA and [3H]aspartate uptake into S1 fractions and slices of hippocampus and cerebral cortex, but not those of caudate nucleus and hypothalamus. Similar inhibition was not observed on using antibodies against synaptosomal membranes of rat caudate nucleus. Anti-HPC IgG raised against synaptosomal membranes of hippocampus failed to alter both spontaneous and K+-evoked release of [3H]noradrenaline. They did not interfere with the binding of [3H]desipramine (the potent noradrenaline-uptake inhibitor) and with the binding of [3H]dihydroalprenolol, thus excluding any interaction of the antibodies with drug receptors which are located on either the pre- or postsynaptic membrane. The anti-HPC IgG inhibit the enzymatic activity of [Na+-K+-]ATPase by 30% upon incubation of the antibodies with crude membrane preparations. A comparison of their inhibitory effects with those of the neurotoxin 6-hydroxydopamine suggests that the corresponding hippocampal specific antigens are located at a presynaptic site.     

Determination of the Equilibrium Dissociation Constants and Number of Glycine Binding Sites in Several Areas of the Rat Central Nervous System, Using a Sodium-Independent System

Parameters affecting the binding of [3H]glycine to membrane fractions isolated from the cerebral cortex, midbrain, cerebellum, medulla oblongata, and spinal cord of the rat were investigated in a Na+-free medium. A [3H]glycine binding assay was established in which the binding was specific, saturable, pH-sensitive, and reversible. Conditions were chosen in an effort to minimize binding to glycine uptake sites. From data on specific [3H]glycine binding Scatchard plots were prepared and the KD and Bmax values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (KD: 44, 211 nM; Bmax: 361, 1076 fmol/mg protein) and spinal cord (KD: 19, 104 nM; Bmax: 105, 486 fmol/mg protein). The ranges of the KD and Bmax values for the other three areas studied were 59 to 144 nM and 882 to 3401 fmol/mg protein, respectively. When the glycine content of each area, expressed as fmol/neuron, was plotted against the respective KD (high affinity), a negative correlation was found (r = --0.90; p less than 0.05). A similar negative correlation was found between the glycine content and Bmax (r = --0.88; p less than 0.05). Hill plots indicated a slope of essentially 1.0 for all areas. GABA, taurine, strychnine, diazepam, bicuculline, and imipramine had little or no effect on [3H]glycine binding.     

Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction

Abstract: Specific binding of tritiated dopamine, spiperone, and N-propylnorapomorphine was examined in subcellular fractions from bovine caudate nucleus. All fractions contained at least two sets of specific binding sites for [³H]spiperone (K_{D 1}^aPP= 0.2 nM, K_{D 2}^aPP= 2.2 nM), the higher affinity sites accounting for one-third to one-eighth of the total. [³H]Spiperone binding was slightly enriched over the total particulate fraction in P₂, P₃, SPM, and a crude fraction of synaptic mitochondria. A microsomal subfraction (P₃B₂) exhibited the highest specific binding capacity obtained, representing a fourfold enrichment over the total particulate fraction. [³H]Dopamine exhibited apparent binding to a single class of high-affinity sites in all fractions examined (K_D^aPP= 4.0 nM). A greater than twofold enrichment was observed in all fractions except myelin and P₃, with a fivefold enrichment in SPM and P₃B₂. At least two classes of receptors were labeled by [³H]-N-propylnorapomorphine (K_{D 1}^aPP= 0.55 nM, K_{D 2}^aPP= 20 nM), using 50 nM-spiperone together with 100 nM-dopamine to define nonspecific binding. Although binding to the higher affinity site was displaced by spiperone, and lower affinity binding by dopamine, comparison of receptor densities with values obtained by using [³H]spiperone and [³H]dopamine directly suggested that [³H]-N-propylnorapomorphine labeled additional sites. We have also examined a postsynaptic membrane (PSM) fraction obtained from SPM by successive extraction with salt and EGTA followed by sonication and separation on a density gradient. [³H]Spiperone binding in PSM was enriched two- to threefold over unfractionated SPM with a concomitant decrease in [³H]dopamine binding. The enrichment in spiperone receptors was almost entirely due to an increase in the number of lower affinity binding sites, suggesting that these sites may be associated with the postsynaptic membrane.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Development and pigmentation of chlorosomes in Chloroflexus aurantiacus strain Ok-70-fl

The development of chlorosomes and their pigmentation were studied by growing Chloroflexus aurantiacus strain Ok-7o-fl first under conditions under which BChl c-synthesis is low (50°C, 2000 lux and 30°C, 1500 lux) and subsequently under conditions promoting high BChl c-synthesis (50°C, 400 lux). Electron microscopic observations on and chemical analyses of isolated cell components showed that in BChl c-depleted cells chlorosome-like structures (chlorosome bags) are attached to fragments of cytoplasmic membranes. These chlorosome bags exhibit a periodic fine structure caused by the construction of the baseplates of the chlorosomes. The baseplates are closely attached to the cytoplasmic membrane, they are rich in phospholipids and apparently contain a 790 nm-BChl a-complex. Chlorosome bags of BChl c-depleted cells always contain a limited amount of light-harvesting pigment complexes (BChlc, - and -carotene). The light-harvesting system is restored (50°C, 400 lux) by first refilling the existing chlorosome bags before cell division takes place.Abbreviations BChl Bacteriochlorophyll - LH Light-harvesting complex - RC Reaction center     

The effects of laminin on the growth and differentiation of embryonal carcinoma cells in defined media

In this paper we have examined the growth and differentiation of the embryonal carcinoma cell line, F₉, in the defined medium EM-3 at low density. We show that the growth of F₉ and their differentiated cells (F₉-diff) in EM-3 is strongly density dependent. At low cell densities the growth of both cell types is severely limited and most of the cells do not survive. Although this poses a problem for working with F₉ and F₉-diff in EM-3, it provides a convenient assay for identifying molecules that support their growth at low density. Using this assay, we have determined that laminin, a newly isolated glycoprotein of basement membranes, significantly improves the growth and short-term survival of both F₉ and F₉-diff. However, addition of laminin to EM-3 is insufficient to promote the clonal growth of these cell types. Our findings also indicate that laminin promotes the attachment of F₉ and F₉-diff in defined media. On the basis of our results, we propose an attachment function for laminin during the early stages of mammalian development.     

Properties of receptors for epidermal growth factor in detergent solution: Evidence for heterogeneous aggregated states

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal forece and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for ¹²⁵I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with ¹²⁵I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble and -insoluble fractions. Gel chromatography of the detergent-soluble membrane fraction on Sepharose 6-B revealed heterogeneity of ¹²⁵I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Stokes radius of 95 Å. Operationally soluble ¹²⁵I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of ¹²⁵I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.     

A structural model for the chromophore-binding domain of ovine rhodopsin

A putative model for the structure of the relatively independent carboxyl-terminal domain of (rhod)opsin has been developed by use of a combination of several secondary structure prediction methods. The validity of this approach was confirmed by comparing the secondary structure for bacteriorhodopsin as predicted by these methods with its known low resolution structure. The resulting predicted structure agreed well with the experimental data. The model obtained for opsin incorporates two transmembrane α-helical rods linked by an intradiscal loop. Each of the helical sections is interrupted by a short irregular region. One of these includes the lysyl residue to which the chromophore 11-cis retinal is attached. The second non-regular segment, almost opposite the first, contains a cysteinyl and a tryptophanyl residue which may be involved in protein—chromophore interaction. The proposed structure of this whole domain could prove instructive in the elucidation of the primary events of visual transduction.