The effect of cholesterol and gramicidin A′ on the carbonyl groups of dimyristoylphosphatidylcholine dispersions

Proton-enhanced carbon-13 magnetic resonance measurements have been made of the natural abundance carbon-13 carbons in hydrated L_α phase dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) codispersed with cholesterol or with the polypeptide gramicidin A′. The carbonyl group spectrum consists of a superposition of two peaks derived from the two carbonyl sites within the lipid. In the L_α phase of DMPC both carbonyl sites contribute axially symmetric spectra, one with a chemical shift anisotropy of –29 ppm and the other with a chemical shift anisotropy of less than –5 ppm. The chemical shift anisotropy of the broader carbonyl resonance was found to increase with increasing cholesterol content. However, in DMPC dispersions with gramicidin A′, the chemical shift anisotropy of the broader carbonyl signal initially increased slightly from that of pure DMPC and then decreased with increasing concentrations of gramicidin A′. The width of the narrower spectral component was essentially unaltered by cholesterol or gramicidin A′. The presence of a narrow component at all concentrations of cholesterol or gramicidin A′ suggests that it is unlikely that any significant conformational changes have occurred at the carbonyl level of the bilayer. We propose that the major effect of cholesterol or gramicidin A′ is to alter the molecular order parameter, S_mol, which reflects the range of angles through which the local molecular long axis of the phospholipid is tumbling.     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.     

N,N-expeditious dialkylation of cyclen (1,4,7,10-tetraazacyclododecane). An astonishing reactivity of cyclen tricarbonyl molybdenum and chromium complexes

After reaction with alkyl iodides and subsequent oxidative removal of the M(CO)₃ triprotection, molybdenum and chromium fac-LM(CO)₃ complexes of cyclen (L) unexpectedly lead to N¹,N⁷-dialkylated cyclen derivatives.     

Kinetics and mechanism of CO substitution of [η-CpFe(CO)3]PF6 with PPh3 in the presence of substituted pyridine N-oxide

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Olefin versus sulfur coordination of benzo[b]thiophene (BT) in Cp(CO)2Re(η:η-μ2-BT)Cr(CO)3

Reactions of Cr(CO)₃(η⁶-BT), in which the Cr is π-coordinated to the benzene ring of benzo[b]thiophene (BT), with Cp′(CO)₂Re(THF), where Cp′ = η⁵-C₅H₅ or η⁵-C₅Me₅, give the products Cp′(CO)₂Re(η²:η⁶-μ₂-BT)Cr(CO)₃ in which the Cr remains coordinated to the benzene ring and Re is bound to the C(2)=C(3) double bond. An X-ray diffraction study of Cp(CO)₂Re(η²:η⁶-μ₂-BT)Cr(CO)₃ (3) provides details of the geometry. This structure contrasts with that of the Cp′(CO)₂Re(BT) complexes that exist as mixtures of isomers in which the BT is coordinated to the Re through either the double bond (2,3-η²) or the sulfur (η¹(S)). Thus, the electron-withdrawing Cr(CO)₃ group in 3 stabilizes the 2,3-η² mode of BT coordination to the Cp′(CO)₂Re fragment. Implications of these results for catalytic hydrodesulfurization of BT are discussed. Crystal data for 3: triclinic, space group

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A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.     

Inactivation of Rabbit Liver Carbonyl Reductase by Phenylglyoxal and 2,3,4-Trinitrobenzenesulfonate Sodium

The chemical modifications of rabbit liver carbonyl reductase (RLCR) with phenylglyoxal (PGO) and 2,3,4-trinitrobenzenesulfonate sodium (TNBS), which are respective chemical modifiers of arginine and lysine residues, were examined. RLCR was rapidly inactivated by these modifiers. Kinetic data for the inactivation demonstrated that each one of arginine and lysine residues is essential for catalytic activity of the enzyme. Furthermore, based on the protective effects of NADP +, NAD + and their constituents against the inactivation of RLCR by PGO and TNBS, we propose the possibility that the functional arginine and lysine residues are located in the coenzyme-binding domain of RLCR and interact with the 2′-phosphate group of NADPH.     

3alpha/beta,20beta-hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH

Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.     

Identification of carbonyl sulfide and sulfur dioxide in porcine coronary artery by gas chromatography/mass spectrometry, possible relevance to EDHF

Incubation of porcine coronary artery rings and cardiac muscle tissue in Krebs buffer followed by GC/MS analysis of the headspace gas revealed two gases, carbonyl sulfide (COS) and sulfur dioxide (SO(2)). The gases were identified by characteristic ions obtained by electron ionization, and by comparison of the retention time on a chromatographic column (GS GasPro) with standards of these gases. Stimulation of the arterial rings with acetylcholine and calcium ionophore A23187 increased the levels of SO(2) and COS in the vascular tissue. We also provide evidence that SO(2) could originate from disproportionation of a very unstable gas, sulfur monoxide (S=O). We suggest potential origins of these gases and discuss their relevance to endothelium-derived hyperpolarizing factor.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.