A possible prebiotic peptide formation from glycinamide and related compounds

We examined an experimental approach to the genesis of protocells in the primeval sea. Glycine polymers with an average chain length of 12 were formed from glycinamide in fluctuating systems (pH 7.2, 80°C, 20 cycles). The resulting glycine polymers gave aggregated leaflet-like structures. A solution of the glycine polymers provided stacked disc-shaped structures in the presence of LiBr and gave sheet structures in the presence of dichloroacetic acid. The shapes of these organized structures were correlated with their molecular structures.     

Role of subunit crosslinking in fibrin solubility.

¹²⁵I-labelled fibrinogen was clotted by thrombin in the presence of activated Factor XIII and the rates of formation of γ dimers and α polymers were measured. These changes in fibrin subunits were correlated with the solubility of fibrin in 1% monochloroacetic acid. In the presence of the factor XIIIa inhibitor, glycine methyl ester, fibrin solubility was found to depend on the level of α polymers formed. A preferential inhibition of α polymer formation rather than γ dimer was observed in the presence of glycine methyl ester.     

A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons.

GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.     

A probable prebiotic peptide formation from glycineamide and related compounds in a neutral aqueous medium participation of nucleoside and 5′-mononucleotide

Summary Several glycine oligomers were formed up to a hexamer in neutral aqueous solution by using Gly-NH₂, an intermediate in the formation of glycine by the Strecker synthesis. The optimum pH was around 7 in order to produce (Gly)₂ in the presence of guanosine. The elongation of the glycine peptide was demonstrated by analyzing the n+1 mer of glycine from the reaction of the Gly-NH₂ and (Gly)_n system. (n=0, 2, 3). The peptide formation was promoted by a basic catalyst such as a nucleic base. Guanosine and GMP showed preferential catalytic effect in regard to peptide formation among nucleosides and 5′-mononucleotides, respectively. This is an example of a specific chemical interaction between an amino acid and nucleic base. Other peptides were formed by using Phac-Phe-NH₂ and β-asparagine. The aqueous mixture of phenylpyruvate, ammonium sulfate and glycine gave a peak corresponding to Phac-Phe-Gly in HPLC. The system including β-asparagine and glycine allowed for the formation of α-Asp-Gly and β-Asp-Gly in a ratio of about 1 to 4.     

Reassembly of wall domains of Roman-snail (Helix pomatia) beta-haemocyanin.

beta-Haemocyanin molecules consist of 20 very large polypeptide chains. These chains are composed of eight structural domains. So-called 'collar' domains can be removed by trypsinolysis of the native cylindrical molecule, resulting in an association of the remaining hollow cylinders into large tubular polymers. Dissociation of the tubular polymers gives one single- and four multi-domain fragments. The role of these fragments in the reassembly process of these tubular polymers was investigated. The two-domain fragment could form tubular polymers. The other domain fragments were not able to form tubular polymers unless in the presence of the two-domain fragment. Tubular polymers with enlarged diameter and ribbon-like structures were observed in the reassembly products when the one-domain fragment was omitted.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Primary structure of protamine from the Northern pike Esox lucius

The basic nuclear protein in the sperm of the Northern pike is a protamine, 32-residues long, which behaved as a single component during ion-exchange chromatography and gel electrophoresis. Amino acid analysis gave close to molar ratios for the eight different residues with no evidence of microheterogeneity. However, the presence of sequence variants was revealed following a combination of automated protein sequencing and cleavage of the protamine by CNBr, endoproteinase Lys-C and thermolysin. At position 28 there is an equal probability of having serine or glycine. At position 9 glycine is found more frequently than serine. The reciprocal nature of the substitutions results in glycine and serine contents which are close to a 4:2 ratio. Pike protamines are homologous to those of the trout but show less sequence variation between components.     

Thermophilic microspheres of peptide-like polymers and silicates formed at 250 degrees C

We examined the possibility of chemical evolution in superheated hydrothermal environments and found the formation of microspheres at 250 degrees C and above from a mixture of glycine, alanine, valine, and aspartic acid. The microspheres did not form at lower temperatures and consisted of silicates and peptide-like polymers that contained imide bonds and amino acid residues having an abundance of valine. The results show the possibility of thermophilic cellular structures, which might be adopted by the extremely thermophilic organisms, if they exist, reported by Baross and Deming.     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

Amino acids derivatives synthesis from nitrogen, carbon and water by electric discharges

Electric discharges between a pair of carbon electrodes were continued for 50 days in a vessel of 5 liters in volume which initially contained nitrogen at a pressure of 15 cm Hg and 200 ml of water. The pressure in the vessel was gradually increased to 60 cm Hg at the end of the run. Gas chromatographic analysis showed that the increase of the pressure mainly results from the production of hydrogen and carbon monoxide. The concentration of ammonia in the aqueous sample was increased to 0.05 M in 50 days of the discharge. After hydrolysis, glycine and serine were detected at the concentrations of 3.4 x 10(-3) M and 0.057 x 10(-3) M in the final solution, respectively, though glycine was found only at the concentration of 6 x 10(-6) M before hydrolysis. TLC analysis indicated the presence of hydantoic acid, N-formylglycine, diketopiperazine, and polymers of glycine.     

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.     

Glycine uptake in brush border vesicles isolated from the rat intestinal cells

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.     

Mechanistic and stereochemical studies on the glycine reductase of Clostridium sticklandii.

Clostridial glycine reductase multienzyme complex which catalyses the reaction: Glycine + ADP + Pi + 2H leads to Acetate + ATP + NH3 was solubilised and fractionated essentially according to the method of Stadtman [T.C. Stadtman (1970) Methods Enzymol. 17A, 956--966] into two components: protein A and 'glycine reductase' fraction. A reconstituted system obtained by combining the two components in the presence of dithiothreitol catalysed the conversion of glycine into acetate concomitant with the phosphorylation of ADP to ATP. Using the reconstituted system, in which the unwanted enzyme activity catalyzing an exchange of the alpha hydrogen atoms of glycine with the protons of the medium had been greatly reduced, it was found that the conversion of (2RS)-[2-14C, 2-3H1]glycine (3H/14C = 7.16) into acetate (3H/14C = 7.03) was attended by the retention of both the C-2 hydrogen atoms of glycine. Conversion of (2S)-[2-2H1, 2-3H1]glycine and (2R)-[2-2H1, 2-3H1]glycine by the reconstituted system gave (2S)-acetate and (2R)-acetate respectively showing that the reductive deamination of glycine occurs through an inversion of configuration. The cumulative information available on the glycine reductase reaction is embodied in a hypothetical mechanism of action for the enzyme.     

Construction of protocellular structures under simulated primitive earth conditions

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins.Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes.Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids.Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.     

Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.     

Solvent effect on hemin-catalyzed polymerization of phenols

Phenol and 4-substituted phenols were polymerized by H₂O₂ as an oxidant and hemin as a catalyst in organic/buffer mixed solvents. The chemical structures of the polymers were similar to those synthesized by the catalysis with horseradish peroxidase. Among the organic solvents used, pyridine gave the highest yield of polymer. Also, the manner of addition of H₂O₂ solution affected the polymer yield.     

Mechanisms of activation,inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core

Excitatory neurotransmission mediated by the N-methyl-D-aspartate subtype of ionotropic glutamate receptors is fundamental to the development and function of the mammalian central nervous system. NMDA receptors require both glycine and glutamate for activation with NR1 and NR2 forming glycine and glutamate sites, respectively. Mechanisms to describe agonist and antagonist binding, and activation and desensitization of NMDA receptors have been hampered by the lack of high-resolution structures. Here, we describe the cocrystal structures of the NR1 S1S2 ligand-binding core with the agonists glycine and D-serine (DS), the partial agonist D-cycloserine (DCS) and the antagonist 5,7-dichlorokynurenic acid (DCKA). The cleft of the S1S2 'clamshell' is open in the presence of the antagonist DCKA and closed in the glycine, DS and DCS complexes. In addition, the NR1 S1S2 structure reveals the fold and interactions of loop 1, a cysteine-rich region implicated in intersubunit allostery.     

Reactivity of 2'-deoxyoxanosine, a novel DNA lesion.

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced from 2'-deoxyguanosine by the reaction with nitrous acid or nitric oxide. We found that dOxo reacted with glycine under physiological conditions. The product was identified by spectrometric data as an adduct between the six membered ring of dOxo and an amino group of glycine. The adduct was more stable than dOxo under physiological conditions. The incubation of an oligodeoxynucleotide containing dOxo with glycine gave also rise to the adduct. These results suggest that dOxo formed in DNA reacts with amino groups of various compounds around DNA in vivo resulting in the adduct.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.