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Chemical proteomics relies primarily on click-chemistry-based protein labeling and biotin-streptavidin enrichment, but these techniques have inherent limitations. Enrichment of intracellular proteins using a totally synthetic host-guest complex is described, overcoming the problem associated with the classical approach. We achieve this by affinity-based protein labeling with a target-specific probe molecule conjugated to a high-affinity guest (suberanilohydroxamic acid-ammonium-adamantane; SAHA-Ad) and then enriching the labeled species using a cucurbit[7]uril bead. This method shows high specificity for labeled molecules in a MDA-MB-231 breast cancer cell lysate. Moreover, this method shows promise for labeling proteins in live cells., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn(2+) ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds α-casein, a multiply phosphorylated protein, with a dissociation constant K(D) of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of α-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of α-casein from solutions of various complexity, including milk and human serum spiked with α-casein. In addition to α-casein, β-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.

Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

The analysis of peptide mixtures from urine and plasma samples using bare (uncapped) SiO2 nanoparticles (NPs) with atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) has been reported. The method was based on the adsorption of positively charged peptides on the surface of negatively charged SiO2 NPs through the electrostatic force of attraction. The adsorption on the surface of SiO2 NPs caused enhancement of ionization efficiency of analytes and subsequently increased the signal intensity of peptides. Maximum signal intensity was obtained at optimized concentration of SiO2 NPs and pH of the aqueous solution. The limits of detection (LODs) obtained for different peptides in deionized water with and without using SiO2 NPs were in the range 4.7-360 nM and 0.1-18.0 microM, respectively. The sensitivity of the proposed method was 21-53-fold better than conventional use of AP-MALDI-MS. In addition, linearity in the range 9.5-95 nM was obtained for the peptide angiotensin-II in deionized water with a correlation of estimation of 0.992 using an internal standard. The proposed method provided a simple way to facilitate the ionization of peptides, reduce sample complexity and increase the tolerance to salts and surfactants in the analysis of biological samples. The applicability of the present method was also demonstrated in the real-world sample analysis of aminothiols and lysozyme using MALDI-time-of-flight (TOF)-MS., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.

Metabolic incorporation of an unnatural amino acid allows researchers to rapidly affinity purify newly synthesized proteins from mammalian cell lysates for direct identification by mass spectrometry.

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.

Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. This study combined the LCM with isotope-coded affinity tag (ICAT) technology and two-dimensional liquid chromatography to investigate the qualitative and quantitative proteomes of hepatocellular carcinoma (HCC). The effects of three different histochemical stains on tissue sections have been compared, and toluidine blue stain was proved as the most suitable stain for LCM followed by proteomic analysis. The solubilized proteins from microdissected HCC and non-HCC hepatocytes were qualitatively and quantitatively analyzed with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) alone or coupled with cleavable ICAT labeling technology. A total of 644 proteins were qualitative identified, and 261 proteins were unambiguously quantitated. These results show that the clinical proteomic method using LCM coupled with ICAT and 2D-LC-MS/MS can carry out not only large-scale but also accurate qualitative and quantitative analysis.

An approach is described for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant-sensitive, was chosen as an experimental model. ICAT-labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected (or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant-sensitive thiol residue of cysteine-283 in creatine kinase, providing proof of principle that an ICAT-based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols.

Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments., (Copyright 2003 Wiley Periodicals, Inc.)

This paper describes a sensitive and selective liquid chromatographic method with fluorescence detection for determination of histamine in brain microdialysis samples from awake rats. Samples containing histamine (10 microl) were derivatized with 20 microl of the reagent consisting of 3 mM 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), 3 mM potassium carbonate and acetonitrile (1:1:18, v/v), thereafter 20 microl volume was injected onto the microbore column packed with C18 silica gel. The histamine derivative contained two pyrene moieties, which generated intramolecular excimer fluorescence (450-540 nm) and allowed clear discrimination from the monomer fluorescence (360-420 nm) emitted by PSE itself. The separation of histamine-pyrene derivative was achieved within 25 min, the detection limit (S/N=3) was 0.3 fmol histamine in 20 microl injected. The basal extracellular levels of histamine collected in 10-min fractions (fmol per 10 microl, mean+/-S.D., not corrected for recovery, n=10 rats) were 35.45+/-4.56 (hypothalamus), 9.05+/-1.56 (prefrontal cortex), 7.83+/-0.86 (hippocampus) and 6.54+/-0.66 (striatum). The voltage-sensitive release of histamine was evaluated by perfusing the probes with high (100 mM) concentration of potassium ions or with sodium channel blocker tetrodotoxin (1 microM), and the calcium-dependent release was tested by perfusion with calcium-free Ringer solution. These data, together with physiologically induced increase of extracellular histamine in four examined brain regions during forced swimming demonstrate that this method is suitable for high-sensitive determination of neuronally released histamine under various pharmacological and physiological conditions.

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

The definition of clean bioprocessing of foods should relate to the discharge of clean effluents that do not disturb functional ecosystems in the environment. Clean effluents should not pollute aquatic or terrestrial environments by increasing the levels or determined bioavailibility of reactive oxygen species (ROS), traces of heavy metals (e.g. arsenic, mercury, lead, cadmium) or radionuclides or other ecotoxicants such as the endocrine disrupting chemicals (e.g. xenoestrogens), herbicides and pesticides. Some saleable foodstuffs can contain very small amounts of potentially toxic components. Strategies dealing with potential toxins should be aimed at targeting remedial bioprocessing to safe limits as stipulated by regulatory agencies, rather than trying to eliminate all toxic components of food so that they can no longer be detected by bioassay or other highly sensitive techniques. The ability to detect even the tiniest amounts of toxicants may not be necessary. This 'overdetection' could lead to inappropriate courses of action in some situations.

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate., (Copyright 2001 Academic Press.)

Background: The Src homology 2 (SH2) domains of cytoplasmic signaling proteins generally bind phosphotyrosine (pTyr) sites in the context of carboxy-terminal residues. SAP (also known as SH2D1A or DSHP), the product of the gene that is mutated in human X-linked lymphoproliferative (XLP) disease, comprises almost exclusively a single SH2 domain, which may modulate T-cell signaling by engaging T-cell co-activators such as SLAM, thereby blocking binding of other signaling proteins that contain SH2 domains. The SAP-SLAM interaction can occur in a phosphorylation-independent manner., Results: To characterize the interaction between SAP and SLAM, we synthesized peptides corresponding to the SAP-binding site at residue Y281 in SLAM. Both phosphorylated and non-phosphorylated versions of an 11-residue SLAM peptide bound SAP, with dissociation constants of 150 nM and 330 nM, respectively. SLAM phosphopeptides that were truncated either at the amino or carboxyl terminus bound with high affinity to SAP, suggesting that the SAP SH2 domain recognizes both amino-terminal and carboxy-terminal sequences relative to the pTyr residue. These results were confirmed by nuclear magnetic resonance (NMR) studies on (15)N- and (13)C-labeled SAP complexed with three SLAM peptides: an amino-terminally truncated phosphopeptide, a carboxy-terminally truncated phosphopeptide and a non-phosphorylated Tyr-containing full-length peptide., Conclusions: The SAP SH2 domain has a unique specificity. Not only does it bind peptides in a phosphorylation-independent manner, it also recognizes a pTyr residue either preceded by amino-terminal residues or followed by carboxy-terminal residues. We propose that the three 'prongs' of a peptide ligand (the amino and carboxyl termini and the pTyr) can engage the SAP SH2 domain, accounting for its unusual properties. These data point to the flexibility of modular protein-interaction domains.

The super high affinity binding sites for [3H]Ro15-4513, a partial inverse agonist of central benzodiazepine receptors, were analyzed in rat hippocampus both in vivo and in vitro. An ultra high sensitive method of autoradiography with an imaging plate system was employed for quantitative analysis of [3H]Ro15-4513 binding. In in vitro binding, the super high affinity binding sites in the hippocampus were observed when the [3H]Ro15-4513 concentration was below 0.5 nM. In vivo, the super high affinity binding sites were only found when the injected dose of Ro15-4513 was below 3.6 microg/kg and almost disappeared when the dose was increased to 10 microg/kg. These results both in vivo and in vitro indicate that there is a significant discrepancy between actual free ligand concentration in vivo and in vitro, and that concentrations in intact brain may be much lower than previously thought.

Regulation of programmed cell death (apoptosis) is crucial for normal development and growth, both prenatally and postnatally. If its role during normal embryogenesis of a given structure is established, a number of related congenital disorders can be explained by a (local) deregulation of apoptosis. In this study, apoptotic cell death patterns during normal development of the murine coronal suture were investigated. Detection of apoptotic cells was undertaken by labeling with Annexin V. Results showed apoptosis occurring at the same time and place as suture initiation. Apoptotic cells are located along the entire established part of the suture and its developing part. Because apoptosis is shown to be highly associated with sutural genesis, the theory of craniosynostosis being the equivalent of deregulation at this locus seems in line with these findings.

The central projections of specific subpopulations of lumbar primary afferents were selectively labeled with the lipophilic tracer DiI in fixed preparations of the chicken embryo. Muscle or cutaneous afferents were selectively labeled by applying DiI to identified peripheral nerves. Medial or lateral afferent populations were selectively labeled by partially lesioning the dorsal root. Muscle and cutaneous afferent populations each contribute to both the medial and the lateral afferent populations. Medial muscle afferents terminate in the intermediate zone and lateral motor column proximally, but only in the intermediate zone distally. Lateral muscle afferents terminate in a ventrolateral region of the dorsal horn both proximally and distally. Medial cutaneous afferents terminate predominantly in lamina III, but a few terminate in the medial region of the intermediate zone. Lateral cutaneous afferents terminate in lamina II and in a ventrolateral region of the dorsal horn. On the basis of the principle termination patterns, specific termination fields were defined and related to the classical cytoarchitectonics of the spinal gray matter. Differential retrograde tracing from the spinal cord with fluorescent dextran-amines demonstrated that the medial afferents originate from the earlier-generated ventrolateral population of large sensory cell bodies, while the lateral afferents originate from the later-generated dorsomedial population of small sensory cell bodies. The medial afferents establish their central projections earlier than the lateral afferents, but for each subpopulation the initial pattern of termination prefigures the mature pattern, throughout the segmental range of the collaterals. Birthdating with 3H-thymidine showed that potential target neurons in the different terminal fields within the dorsal horn are born at different times. In particular, interneurons in lamina II are born after those in lamina III, paralleling the early and late termination of cutaneous afferents in these laminae. Our observations support the notion that primary afferents recognize specific cues in the spinal cord, but also implicate the relative timing of afferent and target differentiation as an important determinant of primary afferent termination patterns.

Methyl farnesoate (MF) binding proteins were identified in the hemolymph of male crabs, Cancer magister, using a tritium-labeled photoaffinity analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [3H]FDK in the presence of increasing amounts of unlabeled MF and the proteins were separated using SDS-PAGE. The associated fluorogram revealed the presence of two specific MF binding proteins with apparent molecular masses of 34 and 44 kDa. MF binding proteins were not detected in other tissues including testes, eyestalks, hepatopancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [3H]FDK from hemolymph MF binding proteins in a dose-dependent way. The apparent dissociation constant (Kd) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand binding assay followed by Scatchard analysis was used to determine a more accurate apparent Kd value of 145 +/- 10 nM. A single MF binding peak was demonstrated when hemolymph samples incubated with [3H]FDK were electrophoresed under nondenaturing conditions.

Purpose: Nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in many cell types, modulates the in vivo disposition of several cytotoxic nucleoside analogs. In this study, a radioligand binding assay was developed for measurement of the NBMPR content of plasma and erythrocytes., Methods: The assay was based on the competition between NBMPR and [3H]NBMPR for high-affinity sites on human erythrocytes membranes. With this assay, we followed in mice changes in the NBMPR content of blood plasma and erythrocytes, following the intraperitoneal injection of the disodium salt of NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR., Results: The radioligand binding assay was able to measure precisely as little as 2.5 pmol of NBMPR, allowing the direct determination of NBMPR concentrations in plasma as low as 16 nM. As few as 8 x 10(3) molecules of NBMPR per cell could be determined in erythrocytes. The NBMPR content of plasma from mice injected with NBMPR-P was maximal at about 20 min after injection and declined to < 0.2% of the peak value by 10 h. Erythrocyte-associated NBMPR was also maximal at 20 min, and declined to 11% of the peak value by 10 h after injection. Time courses for the disappearance of NBMPR from plasma and erythrocytes were monoexponential and yielded half-life values of 0.39 h and 0.68 h, respectively, an apparent volume of distribution of 0.61 l/kg, and a clearance of 1.1 l/h per kg., Conclusions: The radioligand binding assay is a sensitive and facile method for monitoring NBMPR concentrations in mammalian plasma and tissue extracts.

We report here a study of photoaffinity labeling of the V1a-vasopressin receptor with high-affinity, V1-specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([beta,beta-dimethyl-beta-mercaptopropionyl(1), p-azido-Phe2,Val4,Lys8,D-Tyr9] vasopressin), two others containing nitrophenylalanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS-PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non-specific labeling and control experiments with the non-photosensitive analogue produced no labeling at all. Chemical crosslinking of 3H-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1a receptor.

Secretory glial cells in the roof of the last diencephalic prosomer, ependymocytes and hypendymocytes, form the subcommissural organ. The cells of this complex were labelled immunocytochemically, using an antiserum against their specific secretory products. The study aims at the characterization of this cell type in the rat as an anatomical model situation. Radially oriented secretory glial cells remain after birth behind the posterior commissure in the mesencephalic aqueduct. At about postnatal day 10, the cell bodies descend into the conventional ependyma and at postnatal day 25 they assume a compact, rounded appearance. The secretory product they release is involved in the formation of Reissner's fiber. This differentiation in phenotype is not accompanied by a change of the intermediate filament expression. In the adult rat these cells had been labelled immunopositive for cytokeratins 8 and 18 as well as vimentin but not for glial fibrillary acidic protein. DiI-marking from the third ventricle and from the dorsal surface of the brain shows that the basal processes of ependymocytes and hypendymocytes project to the external and internal glial limiting membrane, respectively, through the commissural fiber bundles. Also the subependymal located hypendymocytes have apical processes with contacts to the cerebrospinal fluid. When this secretory cell population is studied with respect to cyto-architectonical changes during ontogeny the results lead to a new understanding of the subcommissural cells. They are not specialized ependymal cells in a regionally restricted and secondary differentiated ependymal area, but rather descendants of an ontogenetically ancient, specific type of radial glia. Characteristic features for all subcommissural cells are that they: (1) appear very early during ontogeny, (2) are derived from a radial oriented glial cell type, (3) carry at least one kinocilium, (4) possess an original intermediate filament pattern, (5) release a secretory product.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.

Two candidate photoaffinity probes are designed from 4-substituted quinazolines known to be potent insecticides/acaricides and NADH:ubiquinone oxidoreductase inhibitors acting at or near the rotenone site. 4-(11-Azidoundecyl-2-amino)quinazoline, based on the undecylamino analog SAN 548A as a prototype, was synthesized in 18% overall yield from ethyl 10-undecenoate by oxidation of the terminal double bond, reductive amination, coupling to 4-chloroquinazoline, and functional group manipulation of the terminal ethyl ester to an alcohol, a mesylate and finally nucleophilic displacement with azide ions. 4-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenethoxy)quinaz oline [the (trifluoromethyl)diazirinyl analog of fenazaquin insecticide/acaricide] was prepared from 4-bromophenethyl alcohol in 31% overall yield by first introducing the trifluoromethylketone moiety followed by its conversion to the (trifluoromethyl)-diazirine and finally coupling to 4-chloroquinazoline as above. Both candidate photoaffinity probes have the inhibitory potency of rotenone (IC50 of 3-4 nM in each case). The azidoundecylamino compound has inadequate photoreactivity whereas that of the (trifluoromethyl)diazirinyl analog is ideal at 350 nm. Radiosynthesis of the latter photoaffinity ligand included introduction of the diazirinyl moiety as the carbene precursor, oxidation of (trifluoromethyl)diazirinylphenethyl alcohol to the corresponding acid with Jones' reagent, and reduction of the phenacetyl chloride intermediate with sodium borotritide to incorporate tritium.

Dye-affinity chromatography is widely and increasingly used for the isolation of various proteins. In particular, the purification of transthyretin can be efficiently achieved by affinity chromatography on immobilized Reactive Yellow 13. Measurement of trace-amounts of dye leaching from affinity columns is important because of possible toxicity or side effects linked with the presence of dye in therapeutic transthyretin preparations. A competitive enzyme immunoassay was developed to monitor yellow-dye column leaching. Biotinylated rabbit anti-Reactive Yellow 13 antibodies (immunoglobulin G fraction) were used as principal reagent. The assay is specific sensitive to 1 ng/ml of Reactive Yellow 13, has a good reproducibility and allows the accurate detection of the dye in the presence of transthyretin.

N-Bromoacetyl-3,3',5-triiodo-L-thyronine and carrier-free [3'-125I]-N-bromoacetyl-3,3',5-triiodo-L-thyronine, to be used for affinity labeling of thyroid hormone receptors, were synthesized using a one-step procedure: a solution of the thyroid hormone 3,3',5-triiodo-L-thyronine and bromoacetyl bromide in ethyl acetate was refluxed for an optimal period of time which depends on the amount of hormone processed. The bromoacetylated hormone thus obtained was then fractionated by high-speed counter-current chromatography which yielded N-bromoacetyl-3,3',5-triiodo-L-thyronine that was pure by the criteria of high-performance liquid chromatography and thin-layer chromatography with different solvent systems. The pure product was well separated from all contaminants including one which in high-performance liquid chromatography was not easily separated from N-bromoacetyl-3,3',5-triiodo-L-thyronine. The latter was characterized by 1H nuclear magnetic resonance, plasma desorption mass spectrometry, thin-layer chromatography, high-performance liquid chromatography, UV spectrophotometry, and melting point. Amounts of 3,3',5-triiodo-L-thyronine ranging from picograms, including carrier-free 125I-labeled triiodothyronine, to 200 to 300 mg can be processed with the equipment used in the present investigation.

The uptake of the alpha-aminocephalosporin cephalexin into brush-border membrane vesicles from rat renal cortex was independent on an inward H+-gradient in contrast to the intestinal transport system. The transport system could be irreversibly inhibited by photoaffinity labeling. Two binding polypeptides for beta-lactam antibiotics and dipeptides with apparent molecular weights 130,000 and 95,000 were identified by photoaffinity labeling with [3H]benzylpenicillin and N-(4-azido[3,5-3H]benzoyl) derivatives of cephalexin and glycyl-L-proline. The uptake of cephalexin and the labeling of the respective binding proteins was inhibited by beta-lactam antibiotics and dipeptides as with intestinal brush-border membranes. These data indicate that the transport systems for beta-lactam antibiotics and dipeptides in the brush-border membrane from rat kidney and small intestine are similar but not identical.

We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and [3H]BSTBA binds to intact duck red cells with a high affinity similar to that of [3H]bumetanide (K 1/2 congruent to 0.1 microM). We incubated duck red cells with [3H]BSTBA, then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0 degree C to prevent dissociation of the [3H]BSTBA. The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H2O2-digested gel slices revealed [3H]BSTBA to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by [3H]BSTBA in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1) Photolabelling of this protein by [3H]BSTBA is blocked when 10 microM unlabelled bumetanide is included in the initial incubation medium with [3H]BSTBA; (2) Photoincorporation of [3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (0.06 microM) similar to that for the reversible, saturable binding of [3H]BSTBA and [3H]bumetanide to duck red cells; and (4) [3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [3H]bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl- in the medium containing the radiolabelled diuretic.