Electron paramagnetic resonance spectroscopic studies of the electron transfer reaction of Hantzsch ester and a pyrylium salt.

The oxidation of Hantzsch ester by a pyrylium cation takes place via electron-proton-electron transfer. The reaction was investigated with EPR spectroscopy using TEMPO and DMPO for inhibition and spin trapping, respectively, of the radicals appearing during the reaction. The present in-depth EPR study of the radical reactions of a NADH analogue indicate a complex electron transfer mechanism in the title reaction.

Measurement of NO in biological samples.

Although the physiological regulatory function of the gasotransmitter NO (a diatomic free radical) was discovered decades ago, NO is still in the frontline research in biomedicine. NO has been implicated in a variety of physiological and pathological processes; therefore, pharmacological modulation of NO levels in various tissues may have significant therapeutic value. NO is generated by NOS in most of cell types and by non-enzymatic reactions. Measurement of NO is technically difficult due to its rapid chemical reactions with a wide range of molecules, such as, for example, free radicals, metals, thiols, etc. Therefore, there are still several contradictory findings on the role of NO in different biological processes. In this review, we briefly discuss the major techniques suitable for measurement of NO (electron paramagnetic resonance, electrochemistry, fluorometry) and its derivatives in biological samples (nitrite/nitrate, NOS, cGMP, nitrosothiols) and discuss the advantages and disadvantages of each method. We conclude that to obtain a meaningful insight into the role of NO and NO modulator compounds in physiological or pathological processes, concomitant assessment of NO synthesis, NO content, as well as molecular targets and reaction products of NO is recommended.

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

Functional significance of the lipid-protein interface in photosynthetic membranes.

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.

Tilt, twist, and coiling in beta-barrel membrane proteins: relation to infrared dichroism.

The x-ray coordinates of beta-barrel transmembrane proteins from the porins superfamily and relatives are used to calculate the mean tilt of the beta-strands and their mean local twist and coiling angles. The 13 proteins examined correspond to beta-barrels with 8 to 22 strands, and shear numbers ranging from 8 to 24. The results are compared with predictions from the model of Murzin, Lesk, and Chothia for symmetrical regular barrels. Good agreement is found for the mean strand tilt, but the twist angles are smaller than those for open beta-sheets and beta-barrels with shorter strands. The model is reparameterised to account for the reduced twist characteristic of long-stranded transmembrane beta-barrels. This produces predictions of both twist and coiling angles that are in agreement with the mean values obtained from the x-ray structures. With the optimized parameters, the model can then be used to determine twist and coiling angles of transmembrane beta-barrels from measurements of the amide band infrared dichroism in oriented membranes. Satisfactory agreement is obtained for OmpF. The strand tilt obtained from the x-ray coordinates, or from the reparameterised model, can be combined with infrared dichroism measurements to obtain information on the orientation of the beta-barrel assembly in the membrane.

Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer.

The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.

Nitric oxide, peroxynitrite and cGMP in atherosclerosis-induced hypertension in rabbits: beneficial effects of cicletanine.

We studied the effect of the furopyridine derivative antihypertensive drug, cicletanine, on blood pressure, vascular nitric oxide (NO) and cyclic guanosine 3':5'-monophosphate (cGMP) content in the aorta and the renal and carotid arteries, aortic superoxide production, and serum nitrotyrosine level in hypertensive/atherosclerotic rabbits. The effect of cicletanine was compared to that of furosemide. Rabbits were fed a normal or a cholesterol-enriched (1.5%) diet over 8 weeks. On the 8th week, the rabbits were treated per os with 2 x 50 mg/kg daily doses of cicletanine, furosemide, or vehicle for 5 days (n = 5-6 in each groups). The cholesterol diet increased mean arterial blood pressure (MABP) from 86 +/- 1 to 94 +/- 2 mm Hg (p < 0.05). Cicletanine decreased MABP in atherosclerotic rabbits to 85 +/- 1 mm Hg (p < 0.05), but it did not affect MABP in normal animals. Furosemide was without effect in both groups. In normal animals, NO content (assessed by electron spin resonance after in vivo spin trapping) in the aorta and the renal and carotid arteries was increased by cicletanine, and the drug increased cGMP in the renal artery as measured by radioimmunoassay. The cholesterol-enriched diet decreased both vascular NO and cGMP and increased aortic superoxide production assessed by lucigenin-enhanced chemiluminescence and serum nitrotyrosine determined by ELISA. In atherosclerotic animals, cicletanine increased NO and cGMP content in the aorta and the renal and carotid arteries and decreased aortic superoxide production and serum nitrotyrosine. Furosemide did not influence these parameters. We conclude that cicletanine lowers blood pressure in hypertensive/atherosclerotic rabbits. The antihypertensive effect of the drug in atherosclerosis may be based on its beneficial effects on the vascular NO-cGMP system and on the formation of reactive oxygen species.

Infrared dichroism from the X-ray structure of bacteriorhodopsin.

A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations.

Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents.

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.

Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labeled lipids in gel-phase membranes.

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.

Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts.

BACKGROUND: The role of NO in the mechanism of preconditioning is not understood. Therefore, we studied the effect of preconditioning and subsequent ischemia/reperfusion on myocardial NO content in the presence of an NO synthase (NOS) inhibitor. METHODS AND RESULTS: Isolated working rat hearts were subjected to preconditioning protocols of 3 intermittent periods of rapid pacing or no-flow ischemia of 5 minutes' duration each followed by a test 30 minutes of global no-flow ischemia and 15 minutes of reperfusion. Test ischemia/reperfusion resulted in a deterioration of myocardial function and a considerable increase in cardiac NO content as assessed by electron spin resonance. Preconditioning improved postischemic myocardial function and markedly decreased test ischemia/reperfusion-induced NO accumulation. In the presence of 4.6 micromol/L N(G)-nitro-L-arginine (LNA), basal cardiac NO content decreased significantly, although test ischemia/reperfusion-induced functional deterioration and NO accumulation were not affected in nonpreconditioned hearts. However, the protective effects of preconditioning on both test ischemia/reperfusion-induced functional depression and NO accumulation were abolished. When 4.6 micromol/L LNA was administered after preconditioning, it failed to block the effect of preconditioning. In the presence of 46 micromol/L LNA, ischemia/reperfusion-induced NO accumulation was significantly decreased and postischemic myocardial function was improved in nonpreconditioned hearts. CONCLUSIONS: Our results show that (1) although NO synthesis by the heart is necessary to trigger classic preconditioning, preconditioning in turn attenuates the accumulation of NO during ischemia/reperfusion, and (2) blockade of ischemia/reperfusion-induced accumulation of cardiac NO by preconditioning or by an appropriate concentration of NOS inhibitor alleviates ischemia/reperfusion injury as demonstrated by enhanced postischemic function.

Direct myocardial anti-ischaemic effect of GTN in both nitrate-tolerant and nontolerant rats: a cyclic GMP-independent activation of KATP.

1. We have recently demonstrated that glyceryl trinitrate (GTN) exerts a direct myocardial anti-ischaemic effect in both GTN-tolerant and nontolerant rats. Here we examined if this effect is mediated by GTN-derived nitric oxide (NO) and involves guanosine 3'5' cyclic monophosphate (cyclic GMP) and ATP-sensitive K+ channels (KATP). 2. Rats were treated with 100 mg kg-1 GTN or vehicle s.c. three times a day for 3 days to induce vascular GTN-tolerance or nontolerance. Isolated working hearts obtained from either GTN-tolerant or nontolerant rats were subjected to 10 min coronary occlusion in the presence of 10-7 M GTN or its solvent. 3. GTN improved myocardial function and reduced lactate dehydrogenase (LDH) release during coronary occlusion in both GTN-tolerant and nontolerant hearts. 4. Cardiac NO content significantly increased after GTN administration in both GTN-tolerant and nontolerant hearts as assessed by electron spin resonance. However, cardiac cyclic GMP content measured by radioimmunoassay was not changed by GTN administration. 5. When hearts from both GTN-tolerant and nontolerant rats were subjected to coronary occlusion in the presence of the KATP-blocker glibenclamide (10-7 M), the drug itself did not affect myocardial function and LDH release, however, it abolished the anti-ischaemic effect of GTN. 6. We conclude that GTN opens KATP via a cyclic GMP-independent mechanism, thereby leading to an anti-ischaemic effect in the heart in both GTN-tolerant and nontolerant rats.

Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy.

The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min.

Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR.

The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.

Lack of correlation between myocardial nitric oxide and cyclic guanosine monophosphate content in both nitrate-tolerant and -nontolerant rats.

We studied the effect of nitroglycerin (NTG) on cardiac nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) content in nitrate-tolerant/nontolerant rats in vivo. The effect of the pharmacological blockade of endogenous NO synthesis and the effect of exogenous NO on cardiac cGMP were also examined. Rats were treated with 100 mg/kg of NTG and corresponding vehicle s.c. three times a day for 2.5 days to induce NTG-tolerance/nontolerance. Rats were then administered a single dose of s.c. 100 mg/kg of NTG to test the effect of NTG in tolerant/nontolerant states, respectively. Nontolerant rats treated with vehicle were controls, and nontolerant rats treated with the NO synthesis inhibitor NG-nitro-L-arginine (LNNA, 20 mg/kg) were negative controls. Another group of nontolerant rats treated i.v. with the direct NO donor sodium nitroprusside (SNP, 3 mg/kg) were positive controls. Cardiac NO assessed by electron spin resonance after in vivo spin-trapping increased 100-fold (P < 0.05) in the positive control, 10-fold (P < 0.05) in the NTG-tolerant group, and 4-fold (P < 0.05) in the single NTG group, when compared to controls. In the negative control group, NO was reduced to near the detection limit (four-fold reduction, P < 0.05). Cardiac cGMP measured by radioimmunoassay was increased significantly (two-fold, P < 0.05) only in the positive control group, and there were no differences among the other groups. This shows that: 1) in vivo cardiac bioconversion of NTG to NO is not impaired in nitrate tolerance; and 2) changes in cardiac NO content are not reflected by changes in cGMP content in nitrate-tolerant and -nontolerant rats.

Spin relaxation measurements using first-harmonic out-of-phase absorption EPR signals.

The dependence on spin-lattice (T1) relaxation of the first-harmonic absorption EPR signal (V'1) detected in phase quadrature with the Zeeman modulation has been investigated both theoretically and experimentally for nitroxide spin labels. Spectral simulations were performed by iterative solution of the Bloch equations that contained explicitly both the modulation and microwave magnetic fields (T. Páli, V. A. Livshits, and D. Marsh, 1996, J. Magn. Reson. B 113, 151-159). It was found that, of the various non-linear EPR displays, the first-harmonic out-of-phase V'1-signal, recorded under conditions of partial saturation of the microwave absorption, is particularly favorable for determining spin-lattice relaxation enhancements because of its superior signal intensity and relative insensitivity to spin-spin (T2) relaxation. By varying the Zeeman modulation frequency it is also possible to tune the optimum sensitivity of the V'1-signal to different ranges of the T1-relaxation time. A Zeeman modulation frequency of 25 kHz appears to be particularly suited to spin label applications. Calibrations are given for the dependence on T1-relaxation time of both the amplitude and the second integral of the V'1-signal recorded under standard conditions. Experiments on different spin labels in solution and in membranes demonstrate the practical usable sensitivity of the V'1-signal, even at modulation frequencies of 25 kHz, and these are used to investigate the dependence on microwave field intensity, in comparison with theoretical predictions. The practicable sensitivity to spin-lattice relaxation enhancements is demonstrated experimentally for a spin-labeled membrane system in the presence of paramagnetic ions. The first-harmonic out-of-phase V'1-signal appears to be the non-linear CW EPR method of choice for determining T1-relaxation enhancements in spin-labeled systems.

Relaxation time determinations by progressive saturation EPR: effects of molecular motion and Zeeman modulation for spin labels.

The EPR spectra of nitroxide spin labels have been simulated as a function of microwave field, H1, taking into account both magnetic field modulation and molecular rotation. It is found that the saturation of the second integral, S, of the first harmonic in-phase absorption spectrum is approximated by that predicted for slow-passage conditions, that is, S approximately H1/1 + PH21, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter, P, depends not only on the T1 and T2 relaxation times, but also on the rate of molecular reorientation and on the modulation frequency. Calibrations for determining the relaxation times are established from the simulations. For a given modulation frequency and molecular reorientation rate, the parameter obtained by fitting the saturation curves is given by 1/P = a + 1/gamma2eT1 . Teff2, where Teff2 is the effective T2. For molecular reorientation frequencies in the range 2 x 10(7)-2 x 10(8) s-1, Teff2 is dominated by the molecular dynamics and is only weakly dependent on the intrinsic T02, allowing a direct estimation of T1. For reorientation frequencies outside this range, the (T1T2) product may be determined from the calibrations. The method is applied to determining relaxation times for spin labels undergoing different rates of rotational reorientation in a variety of environments, including those of biological relevance, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions.

Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a "fluidity gene".

The fluidity of Synechocystis membranes was adjusted in vivo by temperature acclimation, addition of fluidizer agent benzyl alcohol, or catalytic lipid hydrogenation specific to plasma membranes. The reduced membrane physical order in thylakoids obtained by either downshifting growth temperature or administration of benzyl alcohol was paralleled with enhanced thermosensitivity of the photosynthetic membrane. Simultaneously, the stress-sensing system leading to the cellular heat shock (HS) response also has been altered. There was a close correlation between thylakoid fluidity levels, monitored by steady-state 1,6-diphenyl-1,3,5-hexatriene anisotropy, and threshold temperatures required for maximal activation of all of the HS-inducible genes investigated, including dnaK, groESL, cpn60, and hsp17. The causal relationship between the pre-existing thylakoid physical order and temperature set point of both the transcriptional activation and the de novo protein synthesis was the most striking for the 17-kDa HS protein (HSP17) associated mostly with the thylakoid membranes. These findings together with the fact that the in vivo modulation of lipid saturation within cytoplasmic membrane had no effect on HS response suggest that thylakoid acts as a cellular thermometer where thermal stress is sensed and transduced into a cellular signal leading to the activation of HS genes.

Membrane location of spin-labeled M13 major coat protein mutants determined by paramagnetic relaxation agents.

Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.

Effects of Ginkgo biloba extract and preconditioning on the diabetic rat myocardium.

Effects of preconditioning and Ginkgo biloba extract (EGb 761) were studied in isolated nondiabetic and diabetic ischaemic and re-perfused rat hearts. Hearts were randomly divided into five groups in both the age-matched non-diabetic and the 8-week streptozotocin-induced diabetic groups: Group I, hearts were subjected to 30 min of global ischaemia followed by 30 min of re-perfusion; Group II, one cycle of preconditioning consisting of 5 min ischaemia and 10 min re-perfusion before the induction of 30 min of ischaemia and 30 min of re-perfusion; Group III, two cycles of preconditioning; Group IV, three cycles; and Group V, four cycles before the onset of 30 min ischaemia followed by 30 min of re-perfusion. Four cycles of ischaemic preconditioning resulted in a reduction of arrhythmias in non-diabetic rats. Thus, in non-diabetics, the incidence of ventricular fibrillation and tachycardia fell from 92% and 100% (no preconditioning) to 33% (p < 0.05) and 42% (p < 0.05), respectively. Four cycles of preconditioning failed to reduce the incidence of re-perfusion arrhythmias in diabetic subjects. Preconditioning reduced the formation of oxygen free radicals measured by electron spin resonance spectroscopy, but the recovery of cardiac function was low in all non-diabetic and diabetic preconditioned groups. EGb 761 at 25 and 50 mg/kg improved cardiac function in non-preconditioned and preconditioned non-diabetic and diabetic hearts. During re-perfusion in the four-cycle preconditioned non-diabetic and diabetic groups, the amount of free radicals was reduced approximately by 50 and 70% using 25 and 50 mg/kg of EGb 761, respectively. EGb 761 improved cardiac function after ischaemia in both non-preconditioned and preconditioned non-diabetic and diabetic rats. Our data suggest that diabetes could abolish the precondition-induced protection.