A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via formation of an interpenetrating, acid-labile, crosslinked polymer network, that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2-2-Di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions the highly crosslinked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers showed a pH-dependent solubility in THF. Transmission electron microscopy, and dynamic light scattering showed time dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 hours. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance.Phospholipid vesicles are routinely used for intracellular delivery, via fusion with the cell membrane and subsequent release of encapsulated cargo. While useful, vesicle delivery systems suffer from a series of physical limitations, including long-term stability and fusion with other substrates, resulting in inefficient cellular delivery of cargo. Transient stabilization of the phospholipid vesicle membrane may provide for better spatial and temporal control of cargo delivery and release. Whereas, vesicles prepared from naturally-occurring phospholipids offer limited stability in harsh chemical and biological environments, stabilization of the vesicle architecture allows many key advantages to be realized. 1-6 Vesicle stabilization has been achieved via several approaches, including: a) utilizing synthetic, polymerizable phospholipids, and b) polymer scaffolding -partitioning hydrophobic monomers into the vesicle lamella with subsequent polymerization. 1;4;6;7 The resulting stabilized vesicles can be loaded across cellular membranes intact, however, the irreversible stability severely limits cargo release, as well as physiological clearance. Chemically and mechanically robust vesicles that can be controllably destabilized under biologically relevant conditions are more desirable. In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via formation of an interpenetrating, acid-labile, cross-linked polymer network, that can be controllably destabilized and subsequently degraded.Stabilized 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles were prepared by fabricating an acid-cleavable, highly cross-linked copolymer within the hydrophobic lamella of the pre-organized vesicle assembly...

The frequency of oscillation of the Belousov-Zhabotinskii reaction (oxidation of malonic acid by bromate ions in acidified solution containing cerium ions) depends on the initial concentration of reactants, but in each case, a plot of log (frequency) against reciprocal temperature is linear. The calculated activation energies for eight solutions cover a relatively smaIl range. These findings are examined in the light of proposed reaction schemes. Calculations indicate that the frequency is controlled to a significant extent by the rate of reaction between bromide and bromate ions to form HOBr and HBrO,.

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Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, has not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials.