The dimetallic endohedral heterofullerene (EHF), Gd2@C79N, was prepared and isolated in a relatively high yield when compared with the earlier reported heterofullerene, Y2@C79N. Computational (DFT), chemical reactivity, Raman, and electrochemical studies all suggest that the purified Gd2@C79N, with the heterofullerene cage, (C79N)5- has comparable stability with other better known isoelectronic metallofullerene (C80)6- cage species (e.g., Gd3N@C80). These results describe an exceptionally stable paramagnetic molecule with low chemical reactivity with the unpaired electron spin density localized on the internal diatomic gadolinium cluster and not on the heterofullerene cage. EPR studies confirm that the spin state of Gd2@C79N is characterized by a half-integer spin quantum number of S = 15/2. The spin (S = 1/2) on the N atom of the fullerene cage and two octet spins (S = 7/2) of two encapsulated gadoliniums are coupled with each other in a ferromagnetic manner with a small zero-field splitting parameter D. Because the central line of Gd2@C79N is due to the Kramer's doublet with a half-integer spin quantum number of S = 15/2, this relatively sharp line is prominent and the anisotropic nature of the line is weak. Interestingly, in contrast with most Gd3+ ion environments, the central EPR line (g=1.978) is observable even at room temperature in a toluene solution. Finally, we report the first EHF derivative, a diethyl bromomalonate monoadduct of Gd2@C79N, was prepared and isolated via a modified Bingel-Hirsch reaction.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

We have used time-resolved electron paramagnetic resonance (EPR) and quenched-flow kinetics in order to investigate the dynamics of Ca-ATPase conformational changes involved in Ca2+ pumping in sarcoplasmic reticulum (SR) membranes at 2 degrees C. The Ca-ATPase was selectively labeled with an iodoacetamide spin label (IASL), which yields EPR spectra sensitive to enzyme conformational changes during ATP induced enzymatic cycling. The addition of ATP, AMPPCP, CrATP, or ADP decreased the rotational mobility of a fraction of the probes, indicating a distinct protein conformational state corresponding to this probe population, while Pi under conditions producing "backdoor" phosphorylation produced no spectral change. Transient changes in the amplitude of the restricted component associated with the pre-steady state of Ca2+ pumping were detected with 10 ms time resolution after an [ATP] jump produced by laser flash photolysis of caged ATP in the EPR sample. The laser energy was adjusted to generate 100 microM ATP from 1 mM caged ATP. At 0.1 M KCl, the EPR transient consisted of a brief initial lag phase, a monoexponential phase with a rate of 20 s-1, and a decay back to the initial intensity after the ATP had been consumed. Raising [KCl] from 0.1 to 0.4 M slowed the rate of the exponential phase from 20 to 6 s-1. Lowering the pH from 7 to 6, which increased the rate of caged ATP photolysis, eliminated the lag but did not change the apparent rate of the EPR signal rise. Parallel acid quenched-flow experiments conducted at 0.1 M KCl and 100 microM ATP produced fast (50-58 s-1) and slow (20 s-1) phases of phosphoenzyme formation. Increasing [KCl] from 0.1 to 0.4 M decreased the rate of the slow phase of phosphorylation from 20 to 5 s-1, without affecting the fast phase. The close correlation between the slow phase of phosphorylation and the exponential phase of the EPR signal suggests that the spin probe monitors a conformational event associated with phosphoenzyme formation in a population of catalytic sites with delayed kinetics. We propose that this constraint is imposed by conformational coupling between the catalytic subunits in a Ca-ATPase oligomer and that, consequently, the EPR signal reflects changes in quaternary protein structure as well as changes in secondary and tertiary structure associated with ATP-dependent phosphorylation.

Aims: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca 2+ uptake and myofilament Ca 2+ sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) in a redox-dependent manner, improving Ca 2+ handling in isolated myocytes/hearts. dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. Innovation: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. Conclusions: PLN plays a central role in HNOinduced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.

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Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

Hypertension in end-stage renal disease (ESRD) may involve lack of endothelial nitric oxide (NO), as suggested by reduced total NO synthase (NOS) in dialysis patients. One reason might be due to substrate deficiency. To test the hypothesis that uremia is a state of intracellular L-arginine deficiency, uremic plasma was obtained from dialysis patients, and its effect was tested on arginine transport in cultured vascular endothelial cells. L-arginine transport (P < 0.01) was reduced in human dermal microvascular endothelial cells (HDMEC) incubated for 6 h with 20% uremic plasma from peritoneal dialysis and hemodialysis patients obtained immediately predialysis. Similar transport inhibition was seen with ESRD plasma in human glomerular capillary and bovine aortic endothelial cells. Hemodialysis partially reversed inhibition of L-arginine transport. HDMECs incubated for 6 h with synthetic media containing high (uremic) urea concentrations showed inhibition of L-arginine transport, but this was not competitive because acute exposure to urea had no impact on L-arginine transport. Over a 6-h period, urea-induced inhibition of L-arginine transport was not sufficient to inhibit NOS activity, but after 7 days NOS activity was reduced. These cellular findings suggest that substrate delivery may be lowered, thus reducing endothelial NOS activity and contributing to hypertension in ESRD patients.