The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13

Abstract The pCloDF1S encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murein lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF13.     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

The solution structure of ω-Aga-IVB,a P-type calcium channel antagonist from venom of the funnel web spider,Agelenopsis aperta

Summary The 48 amino acid peptides -Aga-IVA and -Aga-IVB are the first agents known to specifically block P-type calcium channels in mammalian brain, thus complementing the existing suite of pharmacological tools used for characterizing calcium channels. These peptides provide a new set of probes for studies aimed at elucidating the structural basis underlying the subtype specificity of calcium channel antagonists. We used 288 NMR-derived constraints in a protocol combining distance geometry and molecular dynamics employing the program DGII, followed by energy minimization with Discover to derive the three-dimensional structure of -Aga-IVB. The toxin consists of a well-defined core region, comprising seven solvent-shielded residues and a well-defined triple-stranded -sheet. Four loop regions have average backbone rms deviations between 0.38 and 1.31 Å, two of which are well-defined type-II -turns. Other structural features include disordered C- and N-termini and several conserved basic amino acids that are clustered on one face of the molecule. The reported structure suggests a possible surface for interaction with the channel. This surface contains amino acids that are identical to those of another known P-type calcium channel antagonist, -Aga-IVA, and is rich in basic residues that may have a role in binding to the anionic sites in the extracellular regions of the calcium channel.Abbreviations TOCSY total correlated spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - COSY correlated spectroscopy     

Dynamic modelling of a helical peptide in solution using NMR data: Multiple conformations and multi-spin effects

Summary Nuclear Overhauser effect (NOE) measurements on molecules in solution provide information about only the ensemble-averaged properties of these molecules. An algorithm is presented that uses a list of NOEs to produce an ensemble of molecules that on average agrees with these NOEs, taking into account the effect of surrounding spins on the buildup of each NOE (spin diffusion). A simplified molecular dynamics simulation on several copies of the molecule in parallel is restrained by forces that are derived directly from differences between calculated and measured NOEs. The algorithm is tested on experimental NOE data of a helical peptide derived from bovine pancreatic trypsin inhibitor.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Immunochemical study on PHI/PHM with use of synthetic peptides

We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.     

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.     

Isolation and characterization of alpha-endorphin and gamma-endorphin from single human pituitary glands

alpha-Endorphin and gamma-endorphin, two closely related peptides of the pro-opiomelanocortin family with characteristic biological activities, were purified to homogeneity from single human pituitary glands and chemically identified. Isolation of the peptides was based on size fractionation by Sephadex G-75 chromatography followed by two HPLC steps using reverse-phase and paired-ion reverse-phase systems and was monitored by radioimmunoassay. During the isolation procedure alpha- and gamma-endorphin-sized material behaved chromatographically and immunologically indistinguishably from synthetic alpha- and gamma-endorphin. The amino acid composition and NH2-terminus of isolated peptides demonstrated their identity as authentic alpha-endorphin and gamma-endorphin. Acetylated forms were absent. In addition, evidence is provided that large forms with alpha- and gamma-endorphin immunoreactivity detected during gel filtration are human lipotropin-(1-74) and -(1-75), respectively. The data substantiate that alpha-endorphin and gamma-endorphin exist as endogenous peptides in the human pituitary gland.     

Two recessive mutations with maternal effect upon colour and cleavage ofXenopus l. laevis eggs

Summary Pale eggs and partial cleavage are two mutations with a maternal effect that are found in the same family ofXenopus l. laevis. The pale eggs have animal hemispheres of a yellow to beige colour and give rise to normally pigmented tadpoles and frogs. The cells of pale embryos contain fewer melanosomes than those of controls. The partial cleavage eggs are characterized by an abnormality of cleavage visible from the eight-cell stage onwards, by abnormal yolk platelet distribution and abnormal cytological features. Cleaved, syncytial and uncleaved areas are observed in these eggs, which are lethal at the blastula stage.