The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

Enzymatic conversion of O-carbamyl-L-serine to pyruvate and ammonia

Treatment of partially hepatectomized male rats with urethan 6 hr after operation resulted in 50–55% inhibition of the incorporation of orotic acid-5-³H into nuclear ribosomal RNA and heterogeneous RNA 18 hr later. Neither partially hepatectomized female rats similarly treated with urethan nor operated male animals treated with an equitoxic dose of butyl carbamate presented evidence of an impairment of nuclear RNA synthesis.     

Spin-labeled polyuridylic acid. Formation of an intermediate structure

Temperature-dependent conformational transitions of spin-labeled poly(U) at low temperature in spermidine and cesium chloride buffer have been measured by electron spin resonance spectroscopy. The Arrhenius plot shows the existence of the order-disorder transition at a temperature close to that obtained from absorbance temperature profiles. However, in addition the formation of an intermediate state is observed during the melting of the ordered poly(U) to its random coil.     

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.     

DEDD regulates degradation of intermediate filaments during apoptosis

Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.     

Formation of an unstable covalent intermediate during the inhibition of electric-eel acetylcholinesterase with 1,3,2-dioxaphosphorinane 2-oxides.

The kinetics of interaction of eel acetylcholinesterase (EC 3.1.1.7) with 1,3,2-dioxaphosphorinane 2-oxides were investigated. It was demonstrated that the rate of spontaneous re-activation as well as the re-activation profile in the presence of 2-pyridine aldoxime methiodide of the inhibited enzyme are irrespective of the leaving group of three inhibitors and exhibit the same values. The dissociation constant of the corresponding Michaelis complex was evaluated by two independent methods and the results were found to be in close agreement. It was shown that the active site is essential for interaction between the enzyme and the various dioxaphosphorinanes. The mixed anhydride of diethyl phosphoric acid and 2-hydroxy-1,3,2-dioxaphosphorinane 2-oxide behaves exactly as would be predicted from a typical diethyl phosphate inhibitor. Enxyme that was incubated with the cyclic acid or the corresponding methyl ester recovered immediately upon extensive dilution. Inhibition of enzyme in the presence of high concentratasions of the corresponding 2-chloro and 2-fluoro derivatives decreased the regeneration rates as well as the maximal amount of the re-activated enzyme. This observation could not be explained in terms of a classical aging process. On the basis of the kinetics observations it is suggested that an unstable covalent phospho-enzyme intermediate is formed during the reaction between acetylcholinesterase and 1,3,2-dioxaphosphorinane 2-oxides.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Discussion of the occurrence of an intermediate hydroxylation.

The last step of the biosynthesis of biotin, i.e. the conversion of dethiobiotin to biotin was studied using E. coli. The three dethiobiotin derivatives hydroxylated at C-2 or C-5 were synthesized and tested as potential precursors of biotin. It appears that none of these compounds is able to support the growth of E. coli C124, a mutant which does not synthesize dethiobiotin, but converts it into biotin. These results strongly disfavour the hypothesis of the activation of the saturated carbons by an hydroxylation process.     

Formation of radicals during heating lysine and glucose in solution with an intermediate water activity

Abstract

Heating glucose with lysine under alkaline conditions (pH 7.0–10.0) was found to take place with consumption of oxygen together with formation of brown-colored compounds. Highly reactive intermediary radicals were detected when lysine and glucose were heated at intermediate water activity at pH 7.0 and 8.0. The detection was based on initial trapping of highly reactive radicals by ethanol followed by spin trapping of 1-hydroxyethylradicals with α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and Electron Spin Resonance (ESR) spectroscopy. The generation of reactive intermediary radicals from the Maillard reactions was favored by enhancing alkaline conditions (pH 8.0) and stimulated by presence of the transition metal ion Fe²⁺. The stability of the nitrone spin traps, N-tert-butyl-α-phenylnitrone and POBN was examined in buffered aqueous solutions within the pH range 1–12, and found to be less temperature dependent at acidic pH compared to alkaline conditions. A low rate (k_obs) of hydrolysis of POBN was found at the used experimental conditions of 70°C and pH 7.0 and 8.0, which made this spin trap method suitable for the detection of radicals in the Maillard reaction system.     

Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules

During adipose conversion of murine 3T3-L1 cells, the arrangement of vimentin intermediate filaments (IFs) changes from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules. The fully developed cage complex surrounding the lipid globule consists of a monolayer of groups of regularly spaced vimentin IFs that in turn is closely ensheathed by a special endoplasmic reticulum cisterna. The same IF cage is also seen in other adipocytes in culture and in tissues. The specificity of the association of lipid globules with vimentin IFs during adipose conversion is discussed as a special form of compartmentalization supporting adipogenesis and is taken as an example of a possible IF function in relation to a cell differentiation process.     

Down-regulation of protein kinase C is due to an increased rate of degradation.

The phorbol-ester-induced loss of protein kinase C that has been documented in many cell types appears to be a critical event in the generation of a cellular refractory state. We have investigated here the synthesis and degradation of the protein kinase C polypeptide in order to determine why its steady-state amounts are depleted in response to phorbol esters. These results indicate that depletion is due to an increased rate of degradation, with no change either in mRNA amounts or in rates of polypeptide synthesis.     

Proline: an essential intermediate in arginine degradation in Saccharomyces cerevisiae.

Results of studies on proline-nonutilizing (Put-) mutants of the yeast Saccharomyces cerevisiae indicate that proline is an essential intermediate in the degradation of arginine. Put- mutants excreted proline when grown on arginine or ornithine as the sole nitrogen source. Yeast cells contained a single enzyme, delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase, which is essential for the complete degradation of both proline and arginine. The sole inducer of this enzyme was found to be proline. P5C dehydrogenase converted P5C to glutamate, but only when the P5C was derived directly from proline. When the P5C was derived from ornithine, it was first converted to proline by the enzyme P5C reductase. Proline was then converted back to P5C and finally to glutamate by the Put enzymes proline oxidase and P5C dehydrogenase.     

Identification of the biochemical degradation pathway of triazophos and its intermediate in Diaphorobacter sp. TPD-1

Triazophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A bacterial strain, Diaphorobacter sp. TPD-1, capable of using triazophos and its intermediate, 1-phenyl-3-hydroxy-1,2,4-triazole (PHT), as its sole carbon sources for growth was isolated from a triazophos-contaminated soil in China. This strain could completely degrade 50 mg l⁻¹ triazophos and PHT to non-detectable level in 24 and 56 h, respectively. During PHT degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) analysis. Using this information, a biochemical degradation pathway of triazophos by Diaphorobacter sp. TPD-1 was proposed. The first step involved in the degradation of triazophos is the hydrolysis of the P–O ester bond of triazophos to form PHT and o,o-diethyl phosphorothioic acid, then the triazol ring of PHT is subsequently cleaved to form (E)-1-formyl-2-phenyldiazene. Subsequently, (E)-1-formyl-2-phenyldiazene is transformed to 2-phenylhydrazinecarboxylic acid by adding one molecular of H₂O. Finally, the carboxyl group of 2-phenylhydrazinecarboxylic acid is decarboxylated to form phenylhydrazine.     

Formation of a covalent intermediate between alpha-chymotryspin and the B-chain of insulin during enzyme-catalyzed hydrolysis.

The reaction between alpha-chymotrypsin (EC 3.3.21.1) and the B-chain of bovine insulin was studied radiochemically, by using the 3 5S-labelled sulfo B-chain. After incubation at pH 8.0, interrupted by the addition of trichloroacetic acid, a radioactive product was isolated from the reaction mixture. The labelled product was eluted in parallel with the enzyme in gel chromatography, and its properties at different H+ concentrations indicated that chemically it was an ester, i.e. a covalent enzyme-substrate intermediate. No interaction between sulfo beta-chain and alpha-chymotrypsinogen or phenyl-methyl sulfonyl fluoride-inhibited alpha-chymotrypsin was obtained during identical conditions.     

Equilibrium constant for conversion of pyruvate to acetyl phosphate and formate

Preparations of pyruvate formate-lyase were made from Escherichia coli cells. Net reversal of the "phosphoroclastic split" of pyruvate was readily demonstrated with these preparations. Incubation of acetyl phosphate with formate resulted in the accumulation of pyruvate in concentrations up to 0.5 mm. Catalytic amounts of coenzyme A were essential. Pyruvate was also readily formed from acetyl coenzyme A and formate. The equilibrium constant of the reaction (pyruvate(-) + HPO(4) (2-) --> acetyl phosphate(2-) + formate(-)) has been determined to be about 23 at 37 C.     

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.