Murray Smigel

Urinary prostaglandins. Identification and origin.

Human urine was analyzed by mass spectrometry for the presence of prostaglandins. Prostaglandin E2 and F2alpha were detected in urine from females by selected ion monitoring of the prostaglandin E2-methylester-methoxime bis-acetate and the prostaglandin F2alpha-methyl ester-Tris-trimethylsilylether derivative. Additional evidence for the presence of prostaglandin F2alpha was obtained by isolating from female urine an amount of this prostaglandin sufficient to yield a complete mass spectrum. The methods utilized permitted quantitative analysis. The origin of urinary prostaglandin was determined by stimulating renal prostaglandin synthesis by arachidonic acid or angiotensin infusion. Arachidonic acid, the precursor of prostaglandin E2, when infused into one renal artery of a dog led to a significant increase in the excretion rate of this prostaglandin. Similarly, infusion of angiotensin II amide led to a significantly increased ipsilateral excretion rate of prostaglandin E2 and F2a in spite of a simultaneous decrease in the creatinine clearance. In man, i.v. infusion of angiotensin also led to an increased urinary eliminiation of prostaglandin E. These results show that urinary prostaglandins may originate from the kidney, indicating that renally synthesized prostaglandins diffuse or are excreted into the tubule. Thus, urinary prostaglandins are a reflection of renal prostaglandin synthesis and have potential as a tool to delineate renal prostaglandin physiology and pathology.

Regional differences in prostacyclin formation by the kidney: Prostacyclin is a major prostaglandin of renal cortex

Microsomes prepared from rabbit renal cortex were found to synthesize substantial amounts of 6-ketoprostaglandin F1alpha from prostaglandin G2 or arachidonic acid during an incubation. In contrast, no 6-ketoprostaglandin F1alpha was formed by renal medullary microsomes which synthesize predominantly prostaglandin E2. Mass spectral confirmation of the structure of 6-ketoprostaglandin F1alpha from these incubations demonstrates the ability of the renal cortex to synthesize prostacyclin.

Characterization and localization of prostaglandin E1 receptors in rat liver plasma membranes

Methodology for measurement and characterization of prostaglandin binding to membranes has been developed. The binding assay was used to study the presence of prostaglandin receptors in high purified cell fractions derived from rat liver. High affinity binding receptors which have a saturation value of 1.0 pmole/mg protein and a dissociation constant of 1.2 nM were found exclusively in the plasma membrane. High affinity receptors were not found in cell fractions containing nuclei, rough microsomes. Golgi complex or mitochondria. The binding by other prostaglandins was competitive with prostaglandin E1. Competitive binding studies were used to obtain dissociation constants for prostaglandins F1α, F2α, B1, B2, A1, A2, and 15-keto prostaglandin E2 which were 1100, 100, 300, 180, 16. 16 and 700 nM, respectively. Eicosa-5.8.11.19-tetraynoic acid, an inhibitor of prostaglandin synthesis did not bind appreciably to the prostaglandin E receptor, whereas two prostaglandin analogues, which have high physiological activity compete effectively with prostaglandin E1 for the receptor. Thus, the binding receptor for the E-type prostaglandins is highly specific both with respect to cell localization as well as the type of substrate. Numerical routines for the fitting of the data and a procedure for the determination of the specific activity of the labelled prostaglandin are provided.

Molecular and applied modulation effects in electron electron double resonance. IV. Stationary ELDOR of very slowly tumbling spin labels

The investigation of very slowly tumbling spin labels by stationary electron electron double resonance (ELDOR) is discussed. When a Zeeman modulation frequency of 270 Hz was employed, spectra which were independent of modulation frequency but not modulation amplitude resulted. Under such conditions, the ELDOR technique permits characterization of rotational processes with correlation times from 10−7 to 10−3 sec even though normal electron spin resonance (ESR) spectra are insensitive to motion in these regions, appearing to be superimposable with the ESR powder pattern of a static random collection of molecules. Quantitative analysis of stationary ELDOR spectra is accomplished employing a density matrix treatment that explicitly includes the interaction of the spins with the applied electromagnetic radiation and Zeeman modulation fields. The effect of molecular motion inducing random modulation of the anisotropic hyperfine and electron Zeeman interactions can be calculated employing either an orthogonal ei...

Very slowly tumbling spin labels: Saturation recovery

Abstract The recovery of electron magnetization following pulsed saturation is determined of spectral diffusion and spin—lattice relaxation processes. For dilute solutions of slowly tumbling spin labels (nitroxide radicals), spectral diffusion arises as the result of rotational diffusion modulating anisotropic electron Zeeman and electron—nuclear hyperfine interactions. The effects of such spectral diffusion upon pulsed microwave experiments (pulsed electron—electron double resonance, saturation recovery employing a Bloch scheme, saturation recovery, and spin echo) are treated quantitavely by employing a form of the stochastic Liouville equation for the spin density matrix appropriately modified to include Markoffian diffusion operators modulating oriental variables and hence anisotropic magnetic interactions. Particular attention is paid to the effects of pulse conditions (pump field intensity, pulse length) and to the details of the isotropic rotational diffusion process (motional model such as brownian, free, or jump diffusion; correlation time).

A competitive protein binding assay specific for prostaglandin E.

Abstract A competitive protein binding assay specific for the measurement of prostaglandin E is described. Cross reactivity with prostaglandin E metabolites is negligible. The sensitivity permits measurement in the low picogram range. The method was applied to measurement of prostaglandin E in platelet rich plasma and urine. It was confirmed that thrombin stimulates prostaglandin E synthesis in platelet rich plasma. Indomethacin reduced urinary excretion rate of prostaglandin in the dog by 90.7%.

[36] Size and shape of membrane protein-detergent complexes: Hydrodynamic studies

Publisher Summary Mild detergents, such as Triton X-100, are effective in solubilizing membrane proteins in largely native conformation. The mechanism of this solubilization is the substitution of bound detergent molecules for previously bound phospholipid/cholesterol molecules, and the result of the process is generally a lipid-free protein–detergent complex. By using sufficient quantities of pure preparations of membrane proteins, traditional hydrodynamic or other physical methods can be used to characterize the sizes and shapes of these complexes. However, when a membrane protein can only be obtained in limited amounts or as a mixture with other proteins, these methods are not applicable. This chapter describes a general method to obtain estimates of the hydrodynamic sizes and shapes of a variety of membrane protein–detergent complexes. These methods can provide information on proteins in crude mixtures if the identification of the species of interest by a biological activity (enzyme activity, ligand, or antibody binding) is possible.

[11] Characterization of prostaglandin E receptor in membranes and its use in the assay of prostaglandin E☆

Publisher Summary Prostaglandins have been implicated in the regulation of a wide variety of physiological activities including the dilation of blood vessels, the metabolism of fat, and the secretion of gastric acids. Some of the effects of prostaglandins seem to be mediated by changes in the level of cyclic AMP. In the animal, the breakdown of prostaglandins is extremely rapid and the basal levels found in tissues are often very low, usually less than 1 μg/g wet tissue.