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At least 559 records · Page 31Linked to original sources
PubMed1992

Light-dependent, covalent immobilization of biomolecules on 'inert' surfaces.

We describe a novel, versatile procedure for the light-dependent immobilization of ligands to 'inert' material surfaces. Covalent immobilization of ligands differing in chemical nature and complexity is accomplished under mild and non-destructive conditions. Topical interaction of ligands with organic or inorganic surfaces is mediated by photoactivable polymers with carbene generating trifluoromethyl-aryl-diazirines which serve as linker molecules. Light activation of aryl-diazirino functions at 350 nm yields highly reactive carbenes, and covalent coupling is achieved by simultaneous carbene insertion into both the ligand and inert surface. Thus, reactive functional groups are not required on either the ligand or the supporting material. These procedures are applicable whenever ligands, from molecules to cells--synthetically or genetically produced, or isolated from biological sources--need to be immobilized for improved performance.

Alkaline Phosphatase↗
PubMed1969

Binding sites of cholinesterases. Alkylation by an aziridinium derivative.

1. Alkylation of acetylcholinesterase and butyrylcholinesterase by 2-chloro-N-(chloroethyl)-N-methyl-2-phenylethylamine was observed. This alkylating agent was more potent than related compounds previously described, and less stable (half-life 8.5min. at 23 degrees ). 2. Alkylation had effects on hydrolysis of substrates varying from activation for indophenyl acetate to inhibition for acetylcholine, and intermediate effects with five other substrates. The effects were on V(max.) and not K(m). 3. Alkylation caused a variety of changes in sensitivity to inhibition by five carbamates, five organophosphates and four other inhibitors, varying from total protection against tetraethylammonium to mildly enhanced sensitivity to urea. 4. The findings suggested the existence of three binding sites, one of which was anionic and another hydrophobic, in addition to the esteratic site.

Acetylcholinesterase↗
PubMed1992

Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP).

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.

Affinity Labels↗
PubMed1997

Evidence for interaction of the fusion protein alpha-SNAP with membrane lipid.

alpha-SNAP [soluble N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein] is required for fusion of transport vesicles with their target membrane. In this study, we have examined the membrane-binding properties of alpha-SNAP. We have found that in several tissues a much larger amount of alpha-SNAP per unit weight of protein is bound to membranes than is free in the cytosol. Biochemical analysis shows that a fraction of alpha-SNAP behaves in ways characteristic of hydrophobic, lipid-associated proteins. These findings suggest that membrane binding may be accounted for, at least in part, by interaction with membrane lipid. Consistent with this idea, binding of newly synthesized alpha-SNAP to brain membranes was found to be independent of functional SNAP receptors and could be accounted for by direct binding of alpha-SNAP to membrane lipid. Furthermore, membrane lipid enhanced the ability of alpha-SNAP to stimulate NSF-dependent ATPase activity.

Adenosine Triphosphatases↗
PubMed1997

Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer.

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

Affinity Labels↗
PubMed2002

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2).

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

Amino Acid Sequence↗
PubMed2003

Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups.

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.

Azirines↗
PubMed2003

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.

Animals↗
PubMed2000

Immobilization of ligand-modified polyamidoamine dendrimer for cultivation of hepatoma cells.

Cationic polyamidoamine dendrimers are known to be highly branched cascade polymers. The core part of these polymers, tris(2-aminoethyl)amine, was immobilized onto polystyrene plates to which animal cells do not adhere. using photoreactive 4-(3-trifluoromethylazirino) benzoyl-N-succinimide (TDBA-OSu). Cells of a rat hepatoma cell line, H4-II-E-C3, adhered to a surface immobilized with a first-generation dendrimer probably through interactions between the terminal amino groups of the dendrimer and the cell membranes. The adhered cells were viable, could proliferate, and exhibited urea synthetic activity. The modification of the terminal amino groups with fructose increased the final number of cells obtained after 5 days of cultivation. Multigeneration dendrimers were prepared by repeated linkage of tris(2-aminoethyl)amine with the amino groups. Theoretically, the number of terminal amino groups available for ligand modification is twice as much for each generation of dendrimer growth. Cells cultivated on multigeneration fructose-modified dendrimers exhibited enhanced urea synthetic activity. The use of ligand-modified dendrimers is, therefore, considered to be very promising for the construction of bioartificial organs based on cultivation of the animal cells.

Animals↗
PubMed1989

Ciamexone suppressed anti-islet ADCC of mononuclear cells and serum from type 1 (insulin-dependent) diabetic patients.

ADCC (Antibody-dependent cellular cytotoxicity) against xenogenic islets has frequently been found in newly diagnosed Type 1 (insulin-dependent) diabetics suggesting that when combined with autologous serum in vitro, the destruction of islet cells caused by mononuclear cells (MNC) reflects islet destruction in vivo. In this study the ability of Ciamexone to suppress the anti-islet ADCC in vitro was investigated. We selected both ADCC positive and ADCC negative subjects from a group of Type 1 diabetics. The targets, islets from neonatal rats, were incubated for one hour with the probands serum. The ADCC was then measured by the 51Cr-release of the serum-treated islets after a 6h-incubation with MNC of the same donor. Results both with and without co-incubation of Ciamexone were compared. Ciamexone does not influence the spontaneous 51Cr-release from neonatal rat islets treated with the probands serum but significantly suppresses the anti-islet ADCC. The fact that Ciamexone suppresses anti-islet ADCC may explain its possible effectiveness in Type 1 diabetes.

Adjuvants, Immunologic↗
PubMed1974

Oxaziridines as possible intermediates in flavin monooxygenases.

The enzymatic hydroxylation reactions of flavin monooxygenases are suggested to involve an oxaziridine as the active oxygenating agent. The chemistry of these monooxygenases and their nonenzymatic model systems are consistent with an oxaziridine intermediate. Several advantages of the oxaziridine model over the previously proposed "carbonyl oxide" and flavin peroxide models are discussed.

Azirines↗