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Biomedical subjects

H Sigrist

Publications and source records attributed to H Sigrist.

49 records · Page 3Linked to original sources
PubMed1977

Intrinsic proteins of the intestinal microvillus membrane. Iodonaphthylazide labeling studies.

Isolated brush border membranes of the intestinal epithelial cell were labeled with a hydrophobic photoactive compound [125U]iodonaphthylazide. High incorporation of the radioactive naphthylazide was noted for molecular weight bands of 99 000, 86 000, 65 000, 54 000 and 30 000. Minimal labeling occurred in the higher bands of 300 000, 135 000, 125 000 and 17 000. The iodonaphthylazide label was not removed by extensive papain digestion whereas chloramine T iodinated membranes released radioactivity under the same conditions. Neither enzymatic nor transport activities were inhibited by the presence of iodonaphthylazide or the irradiation process. On the basis of the presented data it is concluded that the iodonaphthylazide unspecifically labels those portions of membrane proteins which are inserted into the lipid bilayer matrix.

Alkaline Phosphatase↗
PubMed1977

Isolation of a chloroplast N,N'-dicyclohexylcarbodiimide-binding proteolipid, active in proton translocation.

The N,N'-dicyclohexylcarbodiimide-binding proteolipid from lettuce chloroplast membranes has been purified by a novel, rapid technique involving I-butanol extraction and ether precipitation. Reconstitution of this proteolipid into liposomes composed of chloroplast lipids and subsequent incorporation of bacteriorhodopsin resulted in the formation of liposomes exhibiting a light-dependent accumulation of protons. This accumulation was significantly enhanced upon addition of N,N'-dicyclohexylcarbodiimide at concentrations similar to those that inhibit chloroplast adenosinetriphosphatase activity. Radioactively labeled N,N'-dicyclohexylcarbodiimide was found to be incorporated essentially into the proteolipid of the reconstituted liposomes. These results suggest that the functional unit responsible for proton channeling in the chloroplast membrane has been isolated and reconstituted in the native state.

Biological Transport, Active↗
PubMed1975

A hydrophobic form of the small-intestinal sucrase-isomaltase complex.

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.

Amino Acids↗
PubMedDate not supplied

A scanning near-field optical microscope approach to biomolecule patterning.

In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.

Adsorption↗
PubMedDate not supplied

Light-induced coupling of aqueous-soluble proteins to liposomes formed from carbene-generating phospholipids.

A novel bilayer-forming phospholipid analogue with a photoactivatable carbene-generating head group was synthesized and characterized with respect to molecular structure and light-induced reactivity. N'-(1,2-Dimyristoyl-sn-glycero-3-phosphoethyl)-N-[m-[3- (trifluoromethyl)diazirin-3-yl]phenyl]thiourea (PED) was prepared by thiocarbamoylation of synthetic dimyristoylphosphatidylethanolamine with 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine. PED formed liposomes in aqueous media. Gel to liquid-crystalline transitions occurred at 10.5 degrees C. Neither PED- nor PED/dimyristoylphosphatidylcholine mixed liposomes underwent major structural changes when photoactivated. Liposome sizes, determined by electron microscopy, were not altered upon light exposure. PED combines the advantages of facile synthesis and timed carbene reactivity by photoactivation at wavelengths greater than or equal to 320 nm. Conditions used for PED photoactivation did not inactivate catalytically active or complex-forming proteins. Light-induced binding of aqueous-soluble proteins to PED containing liposomes was attained through photoactivation in the presence of myoglobin, streptavidin, or trypsin. The proteins mentioned were utilized to characterize carbene-initiated ligand coupling. Procedures described establish a new and versatile method for the formation of proteoliposomes.

Bacterial Proteins↗
PubMedDate not supplied

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Amino Acid Sequence↗
PubMedDate not supplied

Photoimmobilization of a bioactive laminin fragment and pattern-guided selective neuronal cell attachment.

To attain light-dependent functionalization of biocompatible materials, a photolabel-derivatized, bioactive laminin fragment has been synthesized, chemically characterized, and photoimmobilized. Covalent high-resolution patterning of the laminin fragment CDPGYIGSR to hydroxylated fluorinated ethylene propylene (FEP-OH), poly(vinyl alcohol), and glycophase glass has been achieved. The synthetic peptide CDPGYIGSR was thermochemically coupled to either N-[m-[3-(trifluoromethyl)-diazirin-3-yl]phenyl]-4-maleimidobuty ramide or 4-maleimidobenzophenone. Photolabel-derivatized peptides were radiolabeled, and 20 and 300 microns-sized patterns were visualized by autoradiography. The biospecific interaction of photoimmobilized laminin fragments with cells was investigated by analyzing the selective attachment of NG 108-15 neuroblastoma x glioma cells which bear CDPGYIGSR-specific cell surface receptors. On photopatterned FEP-OH membranes NG 108-15 cells differentiated in serum-supplemented media within 1 day. Specific attachment to the immobilized oligopeptide CDPGYIGSR was assessed in serum-free media with competitive binding studies, showing an 82% decrease in cell adherence after the cell receptors were blocked with soluble CDPGYIGSR.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and characterization of a photoactivatable glycoaryldiazirine for surface glycoengineering.

Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.

Glycosides↗