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At least 37 records · Page 2

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Faguy, David↗

Ultrastructure of the endolymphatic sac in the larva of the japanese red-bellied newt Cynops pyrrhogaster

The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the "floccular" vesicle and the "granular" vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain floccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.

NASA Discipline Neuroscience↗

Towards Co-evolution of Membrane Transport and Metabolism

Protocellular boundaries were inextricably connected to the metabolism they encapsulated: to be inheritable, early metabolism must have led to an increased rate of growth and division of vesicles and, similarly, transport through vesicle boundaries must have supported the evolution of metabolism. Even though explaining how this coupling emerged and evolved in the absence of the complex machinery of modern cells is one of the key issues in studies on the origin of life, little is known about the biochemical and biophysical processes that might have been involved. This gap in our knowledge is a major impediment in efforts to construct scenarios for the origin of life and laboratory models of protocells. A combination of experimental and computational studies carried out by us and our collaborators is aimed at helping to close this gap. Properties of membranes might have contributed to the selection of RNA as an early biopolymer. A kinetic mechanism was proposed (Sacerdote & Szostak, 2005) in which ribose was supplied more quickly than other aldopentoses to primordial cells for preferential incorporation of ribonucleotides into nucleic acids. This proposal is based on a finding that ribose permeates membranes an order of magnitude faster than its diastereomers, arabinose and xylose. Our computer simulations, which yield permeation rates in excellent agreement with experiment, and kinetic modeling explain this phenomenon in terms of inter- and intramolecular interactions involving exocyclic hydroxyl groups attached to carbon atoms of the pyranose ring (Wei and Pohorille, 2009). They also constrain scenarios for the formation of the earliest nucleic acids (Wei and Pohorille, 2013). In one scenario, sugars permeate protocellular walls and subsequently are used to synthesize nucleic acids inside protocells. As long as this process proceeds at the rate faster than 6x10(exp -3)/s, ribose derivatives will be available for synthesis easier than their diastereomers. If nucleosides or their activated derivatives are synthesized outside protocells and subsequently transported across protocellular membranes the kinetic mechanism does not apply because all diastereomers, which have their sugars in the furanose rather than pyranose form, permeate the membrane at approximately the same rate. Properties of membranes might have been also coupled to metabolism involving peptides. Recently, Adamala and Szostak (2013) have shown that a dipeptide inside fatty-acid vesicles catalyzes the formation of another dipeptide that binds to vesicle walls and, by doing so, promotes their growth at the expense of other vesicles. This coupling of metabolism, permeability of vesicles and their growth is the first demonstration of evolutionary advantage imparted by small, membrane-bound peptides. Building on this work we have calculated the rate at which different blocked amino acids are delivered to a protocell for synthesis of dipeptides. We have further shown that the dipeptides are located at the water-membrane interface rather than in the center of the bilayer. On these basis it is anticipated that other dipeptides containing aromatic, but not necessarily hydrophobic amino acids (e.g. tyrosine) could have the same catalytic effects. Insight from these studies allows for estimating the rate of vesicle growth and the rates of dipeptide synthesis required to keep the system in balance. These results, in combination with our earlier studies, lead to a general scenario for evolution from membrane-bound dipeptides to ion channels in the origin of life.

Metabolism↗