OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

Real-Time Analysis of Cellular Response to Small-Molecule Drugs within a Microfluidic Dielectrophoresis Device.

Quantitative detection of the biological properties of living cells is essential for a wide range of purposes, from the understanding of cellular characteristics to the development of novel drugs in nanomedicine. Here, we demonstrate that analysis of cell biological properties within a microfluidic dielectrophoresis device enables quantitative detection of cellular biological properties and simultaneously allows large-scale measurement in a noise-robust and probeless manner. Applying this technique, the static and dynamic biological responses of live B16F10 melanoma cells to the small-molecule drugs such as N-ethylmaleimide (NEM) and [(dihydronindenyl)oxy]alkanoic acid (DIOA) were quantitatively and statistically examined by investigating changes in movement of the cells. Measurement was achieved using subtle variations in dielectrophoresis (DEP) properties of the cells, which were attributed to activation or deactivation of K(+)/Cl(-) cotransporter channels on the cell membrane by the small-molecule drugs, in a microfluidic device. On the basis of quantitative analysis data, we also provide the first report of the shift of the complex permittivity of a cell induced by the small-molecule drugs. In addition, we demonstrate interesting quantifiable parameters including the drug effectiveness coefficient, antagonistic interaction coefficient, kinetic rate, and full width at half-maximum, which corresponded to changes in biological properties of B16F10 cells over time when NEM and DIOA were introduced alone or in combination. Those demonstrated parameters represent very useful tools for evaluating the effect of small-molecule drugs on the biological properties of cells.

FlCRhR/cyclic AMP signaling in myenteric ganglia and calbindin-D28 intrinsic primary afferent neurons involves adenylyl cyclases I, III and IV.

The aims of this study were to improve insight into cAMP signaling in myenteric neurons and glia and identify the adenylyl cyclase (AC) isoforms expressed in myenteric ganglia of the guinea-pig small intestine. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520 nm/580 nm fluorescence emission ratio of the protein kinase A fluorosensor FlCRhR. Forskolin or pituitary adenylyl cyclase activating peptide caused an increase in cAMP levels in cell somas and neurites and elicited a slow EPSP-like response in myenteric AH/Type 2 neurons, whereas the inactive form of forskolin was without these effects. Glia displayed similar cAMP responses. Immunoblot analysis showed that AC I, III and IV were present in myenteric ganglia, with AC I being detected as two bands of 160 kDa and 185 kDa, AC III as two bands near 220 kDa, and AC IV as two bands of greater than 220 kDa. Pretreatment with N-ethylmaleimide and N-glycosidase F revealed an AC IV band at 115 kDa. Preabsorption with specific blocking peptides prevented detection of AC I or AC IV immunoreactive proteins. In ganglia which expressed strong AC IV immunoreactivity, no immunoreactive bands were detected for AC II, AC V/VI, AC VII or AC VIII. The amount of AC isoforms expressed in myenteric ganglia followed the order of AC IV&z.Gt;III>I. Immunofluorescent labeling studies revealed that AC I, AC III and AC IV were variably expressed in myenteric neurons and glia of the duodenum, jejunum and ileum. In the guinea-pig ileum, AC I, III and IV immunoreactivities were respectively present in 26%, 58% and 89% of calbindin-D28-colabeled myenteric neurons. These findings suggest that (1) AC I, AC III and AC IV variably contribute to cAMP signaling in myenteric ganglia, (2) AC I, AC III and AC IV may be differentially expressed in distinct subsets of calbindin-D28 neurons which may represent intrinsic primary afferent myenteric neurons. Our study also provides direct evidence for activation of cAMP-dependent protein kinase.

N-terminal fibronectin 30-kDa fragment mediates the immobilization of soluble fibrin by factor XIIIa-coated polystyrene beads.

Polystyrene beads coated with thrombin-activated Factor XIII (plasma transglutaminase, plasma transamidase) retained soluble 125I-fibrin, if a 30-kDa fragment of fibronectin was present. The fragment was obtained by proteolytic cleavage of plasma fibronectin with trypsin, but was also available with plasmin or thrombin. It represented a fibrin-binding domain at the N-terminus of each of the two subunit chains and contained close to its amino end a transamidase-reactive site. Fibronectin was unable to mediate the binding of 125I-fibrin to Factor XIIIa-coated beads. 125I-fibrinogen was hardly recognized by the beads even in presence of the fibronectin fragment. The relatively slow binding of 125I-fibrin was inhibited by 0.15 mM putrescine or by a pretreatment of the coated beads with EDTA or N-ethylmaleinimide indicating the involvement of a transamidation in the binding reaction. Immobilization of 125I-fibrin in presence of the fibronectin fragment is assumed to require a covalent cross-linking of the two ligands at the immobilized transamidase giving rise to a product which is retained strongly. The possibility is discussed that a surface-attached transamidase might act as a fibrin receptor which requires the fibronectin fragment as a cofactor.

Reaction of fluorescein isothiocyanate with thiol and amino groups of sarcoplasmic ATPase.

Several model compounds containing thiol and/or amino groups (mercaptoethanol, glutathione, cysteine, ethanolamine, glycine) were studied with respect to their reactivity towards fluorescein isothiocyanate (followed spectrophotometrically at 504 and 412 nm), stability of product and long-wave absorption maximum of the fluorescein residue attached. Thiol groups reacted by far more readily than amino groups. A specific effect was observed with cysteine, indicating an intramolecular transfer of the fluorescein residue from SH to NH2. With sarcoplasmic vesicles both types of reactions were observed. The ratio of products, which can be distinguished by their different stabilities and absorption spectra, depended on the absence or presence of detergents. While with native vesicles the NH2 reaction predominated, with vesicles solubilized with sodium dodecylsulfate, octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine the SH reaction became prevailing. Already 0.35 mg sodium dodecylsulfate per mg protein were sufficient to give rise to dithiourethane formation exclusively. Excess fluorescein isothiocyanate reacted with several thiol groups of dodecylsulfate-solubilized vesicles. In the presence of ATP binding of fluorescein isothiocyanate to native vesicles was significantly reduced. Total blockage of the vesicular SH groups with N-ethyl-maleimide led to preparations that reacted with fluorescein isothiocyanate much more slowly, compared to native vesicles. Octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine in the assay accelerated the thioureide formation from N-ethylmaleimide modified vesicles, whereas sodium dodecylsulfate prevented it almost completely. Our results support the suggestion that one or several thiol groups in vicinity of the highly reactive lysyl residue might play a role in the fast specific reaction, which is only observed with intact native vesicles.