Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.     

Cell adhesion and spreading factor. Chemical modification studies

The purified fetal calf serum factor that promotes cell adhesion and spreading of baby hamster kidney cells on tissue culture substrata has been subjected to a variety of chemical modifications and then tested for activity. These studies have shown that modification of the carbohydrate portions of the factor by glycosidic enzymes or by periodate oxidation did not alter its ability to promote cell spreading. On the other hand, modification of some protein portions of the factor by proteolytic enzymes or by specific modification of —COOH groups, tyrosine residues, or tryptophan residues resulted in a marked inhibition of factor activity. Modification of protein —SH groups, —NH₂ groups, or methionine residues did not affect factor activity. Control experiments indicate that the various modifications were directed at the activity of the factor and not its adsorption onto the substrata.     

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Chemical biology studies on norrisolide

The cellular activity of norrisolide (7), a novel Golgi-vesiculating agent, was dissected as function of its chemical structure. This natural product induces irreversible vesiculation of the Golgi membranes and blocks protein transport at the level of the Golgi. The Golgi localization and fragmentation effects of 7 depend on the presence of the perhydroindane core, while the irreversibility of fragmentation depends on the acetyl group of 7. We show that fluorescent derivatives of norrisolide are able to localize to the Golgi apparatus and represent important tools for the study of the Golgi structure and function.