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A 48-kDa [beta]-N-acetylglucosamine (GIcNAc)-binding protein was isolated from mouse brain by GIcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse [Na.sup.+]/[K.sup.+]-ATPase [beta]1-subunit. When the recombinant FLAG-[beta]1-subunit expressed in Sf-9 cells was applied to a GIcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCI, little of the proteins bound to a GIcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCI at concentrations above 1 mM. Immunohistochemical study showed that the [beta]1-subunit and GIcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-[beta]1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the [Na.sup.+]/[K.sup.+]-ATPase [beta]1-subunit is a potassium-dependent lectin that binds to GIcNAc-terminating oligosaccharides: it may be involved in neural cell interactions. GIcNAc-binding lectin | cell surface | mouse brain

The preparation of synthetically useful aldehyde from 3-phenyl-3-trifluoromethyl-3H-diazirine is described, which has enabled many applications, including the catalytic asymmetric synthesis of the photoreactive phenylalanine analog L-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-phenylalanine (TmdPhe). This new method has the potential to make diarizine photophores useful building blocks for the preparation of diverse photoaffinity probes.

A photoaffinity labeling (PAL)-based method for the rapid identification of target proteins is presented in which a high-performance chemical tag, an isotope-coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target-selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins. [ABSTRACT FROM AUTHOR]

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores-aryl azide, benzophenone and trifluoromethyldiazirine- are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing L-phenylalanine derivatives. [ABSTRACT FROM AUTHOR]

The article discusses a study on the effective method for providing optically active diazirinyl homophenylalanine (hPhe) featuring 3-(3-methoxyphenyl)-3-(trifluoromethyl)-3H-diazirin as well as the aspastic acid's β-acid chloride. The study employed photoaffinity labeling and focused on developing the (3-trifluoromethyl) diazirinyl photophore into hPhe. The study found that the Friedel-Crafts reaction in triflic acid afforded derivatives without any loss of the optical purity.

Photochemical mapping of nucleophiles in close proximity to the active site Cys149 of rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was demonstrated based on the nucleophilic aromatic photosubstitution reaction using two regioisomers of alkoxy-fluoro-nitro-substituted benzenes. Two photophores were covalently attached to the active site SH group of GAPDH and the protein was subjected to photolysis then to the cyanogen bromide cleavage reaction. The advantage of this method is the capability to chase labeled products by monitoring absorption at 380 nm because of the chromogenic property of photophore. HPLC separation identified a large labeled peptide fragment that was further digested by V8 protease for Edman sequence analysis. From the recent X-ray crystallography of rabbit GAPDH, Tyr311, His176, Ser238 and Lys183 are closely located to catalytic Cys149. Among these nucleophiles, Tyr311 was preferentially labeled with 2-fluoro-4-nitrophenoxy photophore and no label was identified with the isomeric 4-fluoro-2-nitrophenoxy photophore. The result clearly reflects the distance between Cys149 and nucleophiles to distinguish the nearest Tyr311. As photophores show great reactivity even with water under neutral conditions, the distance between nucleophiles and photophores is important for photo-induced nucleophilic aromatic substitution. The method will provide a useful technique to survey nucleophiles within the catalytic domain. [ABSTRACT FROM AUTHOR]

A 48-kDa β-N-acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GIcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na+/K+-ATPase β1-subunit. When the recombinant FLAG-β1 -subunit expressed in Sf-9 cells was applied to a GIcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCI, little of the proteins bound to a GIcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCI at concentrations above 1 mM. immunohistochemical study showed that the β1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. inclusion of anti-β1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na+/K+-ATPase flu- subunit is a potassium-dependent lectin that binds to GIcNAc- terminating oligosaccharides: it may be involved in neural cell interactions. [ABSTRACT FROM AUTHOR]

An efficient synthesis of tag introducible (3-trifluoromethyl)phenyldiazirine based phenylalanine derivatives is described. Alkylation of a chiral glycine equivalent with a spacer containing (3-trifluoromethyl)phenyldiazirinyl bromides enables us to create photoreactive l-phenylalanine derivatives. After introduction of biotin at the spacer, the biotinylated and photoreactive amino acid was applied for l-amino acid oxidase and incorporated into a substrate binding site. These compounds will be powerful tools not only for photoaffinity labeling to elucidate properties of bioactive peptides but also as trifunctional photophors to introduce a ligand skeleton. [Copyright &y& Elsevier]

We describe a rapid HPLC method for measuring the isomerization levels of Asp-residues within peptides. This method can detect the isomerization of Asp-residue at a level of less than 1% without performance of acid hydrolysis. Using this method, we further studied the isomerization of Asp-residues in the αA-crystallin f(55–65) fragment of sequence TVLDSGISEVR (T6). Four isomers of T6 peptides, which contain L-Asp, L-isoAsp, D-Asp, and D-isoAsp, respectively, were synthesized. Following incubation of any one of these peptides, four possible isomers of T6 peptides were found in an aqueous buffer. The isoAsp/ Asp ratios of peptides in these solutions were found to reach 3.6–3.8 after incubation at 90°C for 72 hr. [ABSTRACT FROM AUTHOR]