Tuvia Bercovici

Intrinsic proteins of the intestinal microvillus membrane. Iodonaphythylazide 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.

The selective detection of cell surface determinants by means of antibodies and acetylated avidin attached to highly fluorescent polymer microspheres.

Procedures are described for the synthesis of 500 A-diameter polymer microspheres containing a novel fluorescent cross-linking agent. These microspheres have very high fluorophore concentration without quenching of the fluorescence and show very low nonspecific interaction with cells. When monoclonal anti-Thy-1.2 is attached to the fluorescent microspheres, specific binding results in 10(4) spheres being attached per thymocyte while non-specific binding is less than 1%. Similar values are obtained for an indirect staining procedure. The high non-specific binding of cationic avidin to negative cell surfaces is shown to be decreased to negligible levels by acetylation of the amine groups of the protein without decreasing its high-affinity binding to biotin. The use of acetyl-avidin (pI = 6.7) directly, or when attached to fluorescent microspheres, resulted in a highly selective detection of biotinyl groups on the erythrocyte or lymphocyte cell surface. Attachment of biotinyl groups to the hinge carbohydrates of antibodies did not affect their specificity. It allowed their detection by means of microspheres-acetyl-avidin conjugates.

Membrane insertion of lymphocyte surface molecules.

We have used different radiolabelling procedures, followed by immunoprecipitation and SDS-PAGE†, to assess the membrane disposition of various lymphocyte surface molecules. [125I]-lodonaphthylazide labels lipid-associated portions of membrane molecules, whereas lactoperoxidase-catalysed iodination labels protein segments which are exposed on the outside of the plasma membrane. Our results here show that in murine splenocytes, surface Ig μ and δ heavy chains, H-2 heavy chains, and I-A alpha and beta chains are all inserted into the lipid bilayer and exposed on the cell surface. Surface Ig L chains and H-2-associated beta-2-microglobulins are not membrane-inserted, but are bound to the cell surface via association with their respective heavy chains. I-A invariant chains are labelled neither on the cell surface nor within the lipid bilayer, and so may reside in the cytoplasm. With surface Ig δ chains, the Fc rather than the Fd portion is shown to be membrane-inserted. Finally, an unidentified 28K dalton protein, also residing within the lipid bilayer, is shown to be coprecipitated with surface Ig.

The role of conformers in the reversible photocyclisation of cis-1,2-diarylethylenes. A flash-photolytic study

Flash-photolytic experiments showed that the photocyclisation of 1,2-di(2-naphthyl)ethylene, (Ia), results in the formation of two isomeric 4a,4b-dihydro-phenanthrenes derived from two of the possible three conformers of (Ia), and differing in thermal stability by a factor of 1010.

The photochemistry of di-p-cumenylfulgide (bis-p-cumenylmethylenesuccinic anhydride)

The photochemistry of the title compound in the solid and in solution at low temperatures has been investigated. The solid E,E-isomer gives the E,Z-isomer plus a dimer F; one crystal form of the E,E-isomer is reversed photochromic. The solid Z,Z-isomer also gives the dimer F, whereas the E,Z-isomer appears to give the E,E-isomer as the primary photoproduct. In methylcyclohexane–isohexane at –185°, the E,E-isomer is isomerised to E,Z; the latter is reversibly isomerised into Z,Z but also gives two dimers, one of them F. At –100° the same isomerisation processes occur, with in addition reversible conversion of the E,Z-isomer into the 1,8a-dihydrophenylnaphthalene. At these low temperatures the isomerisation of the E,E- to the Z,Z-isomer proceeds via the E,Z-form. The isomerisation processes do not proceed through the dihydrophenylnaphthalenes or cyclobutenes.