Albumin catalyzes the transformation of prostaglandin D2 to 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and to isomeric prostaglandin D2 compounds including delta 12(E)-prostaglandin D2. Both of these compounds are alpha,beta-unsaturated ketones, which should render them susceptible to nucleophilic addition. We therefore examined the ability of the compounds to form conjugates with thiols glutathione and cysteine. During incubation with excess glutathione, both 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2 formed a conjugate. Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 occurred very rapidly; approximately 70% was conjugated within 2 min. In contrast, conjugation of delta 12(E)-prostaglandin D2 with glutathione proceeded at a much slower rate; only 38% was conjugated at 60 min. The formation of both conjugates was enhanced by glutathione S-transferase. Conjugation of both compounds with cysteine was found to occur more rapidly than with glutathione. This effect was more pronounced with delta 12(E)-prostaglandin D2 in which 60% conjugated with cysteine within 2 min. These differences are likely attributed to greater steric hindrance for conjugation across the delta 12 double bond compared to that across the delta 9 bond. Analysis by fast atom bombardment mass spectrometry confirmed the formation of the glutathione conjugate of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2. Following prolonged incubation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with excess glutathione in the presence of glutathione S-transferase, a small quantity of a bis conjugate of this compound was also detected by mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1990

Monomer sequence determination of carbohydrates using fast-atom bombardment mass spectrometry of periodate-oxidized acetate ester derivatives.

A derivatization method, adapted from that of Angel et al. (ref. 10), for sequencing sugar residues in partially degraded poly- and oligo-saccharides using positive-ion f.a.b.-m.s. is described. Derivative selection provides sequence information by directing fragmentation exclusively to both sides of glycosidic oxygen atoms and, in the case of opened rings, between glycosidic carbon and ring oxygen atoms. Polysaccharides or oligosaccharides are subjected to sequential periodate oxidation, borodeuteride reduction, and acetylation. The derivatized polysaccharides are then subjected to partial degradation, acetylation, and high-performance liquid chromatography (h.p.l.c.) purification. F.a.b.-m.s. data obtained on model compounds, using 3-nitrobenzyl alcohol as matrix for f.a.b.-m.s., demonstrated direction of fragmentation to both sides of the glycosidic oxygen atom in unoxidized residues, and to both sides of the acetal oxygen atoms in oxidized residues. Oligosaccharide linkage and sequence may thus be determined by observing fragmentation from both the reducing and non-reducing ends of the molecule. Two Salmonella lipopolysaccharides, derivatized by this procedure, were partially hydrolyzed and then acetylated. Analysis of the h.p.l.c.-purified oligosaccharide derivatives by f.a.b.-m.s. demonstrated the applicability of the technique for sequencing nmol quantities of branched structures.

Benzyl Alcohols↗

PubMed1990

Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with intracellular glutathione and enhancement of its antiproliferative activity by glutathione depletion.

The major dehydration product of prostaglandin D2, 9-deoxy-delta 9,delta 12(E)-prostaglandin D2, is a potent cytotoxic compound. Like other cytotoxic prostaglandins, this compound possesses an alpha, beta-unsaturated ketone group to which cytotoxic activity has been attributed. This prostaglandin was found to readily conjugate with glutathione (GSH) in vitro. When 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 was incubated with Chinese hamster ovary or hepatoma tissue culture cells, it was rapidly taken up and was recovered in the cell lysate primarily as a GSH conjugate in which the keto group at C-11 and the delta 12 double bond had been reduced. Identification of the GSH conjugate was accomplished by analysis by fast atom bombardment mass spectrometry following purification by high performance liquid chromatography. This GSH conjugate and its cysteinylglycinyl and cysteinyl metabolites were also identified in the cell culture medium. 9-Deoxy-delta 9,delta 12(E)-prostaglandin D2 inhibited cell proliferation of these two cell lines in a concentration dependent manner. Depletion of intracellular glutathione by treatment with diethyl maleate and buthionine sulfoximine decreased the amount of intracellular conjugated prostaglandin recovered, and significantly enhanced the antiproliferative effect of 9-deoxy-delta 9-delta 12(E)-prostaglandin D2 on the growth of these cell lines in a concentration dependent fashion. We conclude that intracellular GSH may modulate the antiproliferative activity of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and, possibly, of other cytotoxic prostaglandins.

Animals↗

PubMed1990

Mass spectrometry of carbohydrates.

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Carbohydrates↗

PubMed1989

Bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate: synthesis, characterization, and reaction with the anion-exchange channel in intact human erythrocytes.

We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Preparation of omega trideuterated sulfidopeptide leukotrienes: analysis by FAB/MS.

C-20 Trideuterated leukotriene A4 methyl ester was prepared by Wittig condensation of a deuterated C9-phosphonium salt with a C11-epoxydienal. It was then treated separately with glutathione, cysteinylglycine and cysteine. The resulting esters were saponified to give C-20 trideuterated leukotrienes C4, D4 and E4 respectively. Fast atom bombardment mass spectrometry showed that the synthetic compounds contained from 0.5 to 1.0% protium impurity. Introduction of deuterium at C-20 ensures that it is stable to a wide variety of reagents and reaction conditions. The deuterated leukotrienes will serve as internal standards for the quantitative analysis by mass spectrometry of endogenous sulfidopeptide leukotrienes present in biological fluids.

Deuterium↗

PubMed1988

Bis(sulfo-N-succinimidyl) [15N,2H16]doxyl-2-spiro-4'-pimelate, a stable isotope-substituted, membrane-impermeant bifunctional spin label for studies of the dynamics of membrane proteins: application to the anion-exchange channel in intact human erythrocytes.

We have synthesized and characterized an isotopically substituted homologue of the membrane-impermeant bifunctional spin label bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP) [Beth et al. (1986) Biochemistry 25, 3824-3832] in which the nitroxide N is substituted with 15N and all of the protons in the doxylpimelate moiety are replaced by deuterons ([15N,2H16]BSSDP). Like its normal isotope homologue, [15N,2H16]BSSDP reacts with the anion-exchange channel in intact human erythrocytes at a site that spans the single extracytoplasmic chymotryptic cleavage site and that overlaps the stilbenedisulfonate site. The narrower line widths in the EPR spectrum of [15N,2H16]BSDP-labeled anion channels allow calculation of a minimum separation of 16 A between spin labels bound at the functionally important stilbenedisulfonate sites on adjacent subunits of an anion channel dimer. The 15N and 2H isotopic substitutions also provide substantial improvement in signal to noise of motionally sensitive regions of the ST-EPR spectrum of [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes. [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes were cross-linked to covalent dimers in the extracytoplasmic domain with the membrane-impermeant cross-linking reagent bis(sulfo-N-succinimidyl) suberate [Staros (1982) Biochemistry 21, 3950-3955], and the saturation-transfer EPR spectrum of these cells was compared with that of cells treated with [15N,2H16]BSSDP but not subsequently cross-linked. The spectra were essentially identical, supporting the hypothesis that anion channel subunits form stable dimers in the membranes of intact erythrocytes.

Anions↗

PubMed1988

3-Nitrobenzyl alcohol has wide applicability as a matrix for FAB-MS.

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Benzyl Alcohols↗

PubMed1988

Depletion of brainstem epinephrine stores by alpha-methyldopa: possible relation to attenuated sympathetic outflow.

The antihypertensive effect of alpha-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr X 12 days), significantly increased alpha-methylnorepinephrine (MNE) in brain (1.02 +/- 0.33 micrograms/g), heart (1.67 +/- 0.57 micrograms/g) and adrenal glands (114.93 +/- 50.47 micrograms/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 +/- 0.44 micrograms/g (n = 6) in the adrenal gland but only 99 +/- 26 pg/g (n = 3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME. We conclude (1) although ME is a metabolite of MD, it is present in extremely low concentrations in brainstem and (2) central epinephrine-containing neurons are depleted of neurotransmitter by MD therapy. If this selective epinephrine depletion occurs in the bulbospinal tract neurons responsible for maintaining sympathetic tone, then this effect could contribute to the antihypertensive effect of MD.

Adrenal Glands↗

PubMed1987

Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+, K+-ATPase inhibitors in acutely volume-expanded hog.

Na+, K+-ATPase inhibitors possessing inhibitory activities against the specific binding of ouabain to Na+, K+-ATPase and 86Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as gamma-arachidoyl- [LPCA(gamma), 34%], beta-arachidoyl- [LPCA(beta), 4%], gamma-linoleoyl- (LPCL, 33%), and gamma-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of gamma-docosapentaenoyl-, gamma-eicosatrienoyl-, and gamma-palmitoyllysophosphatidylcholine were also detected by both FAB mass and 1H NMR spectrometric studies. Only gamma-acyl-LPC's showed inhibitory activities on Na+,K+-ATPase and ouabain-binding activities. These LPC's were effective at 100 microM levels in attaining 50% inhibition of the enzyme activity. The inhibition of Na+,K+-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest the gamma-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na+, K+ -ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme.

Animals↗