The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes.

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in Xenopus oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.

Formation of thiol conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2.

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)

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.

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.

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.