Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.

Software using maximum entropy (MaxEnt) analysis has been developed, and used to deconvolute complete electrospray spectra of protein mixtures. It automatically produces zero-charge mass spectra on a molecular mass scale, along with probabilistic quantification so that the reliability of features in the spectrum can be ascertained. Because maximum entropy is faithful to the experimental data, the results tend to have improved resolution and signal-to-noise ratio. This improved performance, particularly regarding resolution, is demonstrated on a haemoglobin containing two &globins separated by 12 Da at mlz 15 867 (0.08°/0). A separation of 12 Da was previously the closest at which mass measurement of two globins was practicable. Also, two hitherto unresolved P-globins from a second haemoglobin, separated by 9 Da (0.06%) were resolved by MaxEnt and their masses accurately measured. These are the first results using rigorous MaxEnt analysis in electrospray mass spectrometry.In the form initially produced by the mass spectrometer, the electrospray spectra of protein mixtures are complex, each protein in the mixture being represented by a series of multiply charged ions on a madcharge ratio scale.Generally these ions occur with mass-to-charge ratio ( M + z H ) l z , where M is the molecular mass of the protein, H' is the mass of the proton (1.00794 u) and z is the number of charges on an ion, a series of consecutive integers. A 15 kDa protein typically produces about 10 peaks with a range of z from 10-20. Larger proteins, e.g., albumins (66kDa) often have 20 or more peaks in the series.To aid interpretation, some means is required for generating a zero-charge mass spectrum, whereby each component in the mixture is transformed from the multiply charged ion series on a masslcharge ratio scale to a single peak on a molecular mass scale. Early methods] tended to produce artefacts and a baseline which increased with mass. A more recent approach2 has achieved an artefact-free zero-charge spectrum with an improved signal-to-noise ratio, but requires prior identification of the charge states in the multiply charged ion series. Current software allows semiautomatic identification of the charge states, but nevertheless a degree of operator intervention is usually required. Moreover, since the data in the original multiply charged spectrum are directly transformed onto a true molecular mass scale, components which are unresolved in the original data remain unresolved after transformation to the zero-charge spectrum. Yet the signals are known to be broadened, by both the isotopic distribution of the elements in the molecule (isotopic broadening) and the mass spectrometer. Hence the true underlying spectrum of masses will be sharper than the peaks in the original data. By incorAuthor to whom correspondence should be addressed. porating this broadening into the program, MaxEnt is able to deconvolute it from the data, thus enhancing the resolution which can be observed in the final MaxEnt mass spectrum. Peak heights grow proportiona...

ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.

The maximum entropy (MaxEnt) technique has been well established in image processing for the last decade. In more recent times MaxEnt has been applied to spectroscopic techniques and shows considerable promise. As part of a much wider evaluation of the Cambridge software, specifically MemSys3, the MaxEnt technique has been successfully applied to electrospray mass spectra.

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