Xanthine oxidase-catalyzed crosslinking of cell membrane proteins

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.     

cis-Diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II (cis-DDP), in HeLa-S3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S1, G2, M, and then into G1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.35 M NaCl extract or residue of HeLa S-phase nuclei revealed that several antigens changed their DNA-crosslinking pattern during the progression of HeLa cells through their reproductive cycle.     

Effect of concanavalin A on the sensitivity of mouse L cell surface glycoproteins to proteolysis

Binding of Concanavalin A to the outer membrane of mouse L cells is found to result in the protection of cell surface glycoproteins from proteolytic digestion by trypsin. Complete sensitivity to proteolysis, however, is restored after removal of bound Con A from the cells.     

Migration of cell surface concanavalin A receptors in pulsed electric fields.

Concanavalin A (con A) receptors on the surface of cultured Xenopus myoblasts redistributed in response to monopolar, pulsed electric fields. The prefield uniform distribution of the receptors became asymmetrical, and was polarized toward the cathodal pole, in the same way as in DC fields. The extent of asymmetry depended on the duration of field exposure, pulse width (or alternatively, interpulse interval), frequency, and intensity. This relationship was most conveniently expressed by using duty cycle, a quantity determined by both pulse width and frequency. Pulses of average intensity 1.5 V/cm induced detectable asymmetry within 5 min. At the lowest average field intensity used, 0.8 V/cm, significant asymmetry was detected at 150 min. For pulses of high duty cycle (greater than 25%), steady state was reached after 30 min exposure and the steady state asymmetry was dependent on average field intensity. For low duty cycle fields, the time required to reach steady state was prolonged (greater than 50 min). Before reaching a steady state, effectiveness of the pulses, as compared with DC fields of equivalent intensity, was a function of duty cycle. A low duty cycle field (fixed number of pulses at low frequency or long interpulse interval) was less effective than high duty cycle fields or DC.     

Susceptible and non susceptible phenotypes of MOPC 173 plasmocytoma to the killing effect of concanavalin A : study on the concanavalin A binding sites

Several cell lines have been isolated in culture from the murine plasmocytoma MOPC 173. They differ in their susceptibility to the agglutination and the killing effect of Concanavalin A. It is shown that for five different cell lines the number of Con A binding sites per surface-unit and the affinity for the lectin are of the same order of magnitude. It is concluded that there is no relationship between the number and affinity of the binding sites and cell killing.     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the 1–220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Specificity and valency of concanavalin A in neuron-substratum adhesion and redistribution of cell surface receptors

Neurons seeded in culture as spherical cells flatten partially to form lamellipodia by which they adhere to the substratum. Lamellipodium formation is stimulated specifically by concanavalin A (Con A) and other mannose-binding lectins in several types of neuronal cells, but not in similarly treated fibroblasts. Conditions that block much of the adsorption of Con A to the substratum have no effect on stimulation of lamellipodium formation by Con A. This suggests that Con A acts in solution on neurons and does not directly bind them to their substrata. Succinylated-Con A (bivalent) binds to the same receptors as native Con A (tetravalent) but does not elicit lamellipodium extension unless crosslinked with anti-Con A IgG. Treatment of neurons with Con A produces local changes in the composition of the cell surface resulting from redistribution of lectin receptor complexes. This redistribution is not as great with SCon A and, like lamellipodium formation, is sensitive to the valency of Con A. A variety of treatments (4 degrees C, trifluoperazine, nordihydroguaiaretic acid, 4-bromphenacyl bromide, and cytochalasin D), inhibit both Con A-receptor redistribution and lamellipodium extension by neurons. Other treatments (colchicine and cycloheximide) prevented neither lamellipodium formation nor redistribution.     

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells.

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the I-220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Changes in cell surface glycoproteins on non-differentiating L6 rat myoblasts selected for resistance to concanavalin A

Four independently selected conA-resistant, non-differentiating rat L6 myoblast cell lines and their parental wild-type populations were examined for cell surface alterations. [³H]conA-binding studies indicated that the variant myoblasts bound significantly less lectin than wild-type cells at 4 and at 37 °C. Scatchard analysis revealed two general types of binding sites (high and low affinity sites) on wild-type cells; the variants appeared to be deficient in the high affinity sites. These changes in conA binding probably play an important role in determining the conA-resistant phenotype. Lectin-binding results could be significantly modified by altering the composition of the serum in the growth medium used to culture myoblasts prior to performing binding experiments, suggesting the existence of productive and non-productive lectin-binding sites on the cell surface. SDS slab gel electrophoresis of [³H]mannose-labelled surface membranes prepared from variant and wild-type cells showed that several glycoproteins of the conA-resistant myoblasts were defective in mannosylation. The conA-binding abilities of a pronase digest of one of these altered regions from variant separations, with a molecular weight of 44 500 D, was found to contain glycopeptides with reduced affinity for the lectin, supporting the idea that variant membranes are deficient in a set of high affinity lectin-binding sites. Studies on [GDP-¹⁴C]-mannose incorporation into lipid by membranes from variant and wild-type myoblasts indicated that the biosynthetic lesion likely involved a mannosyl transferase enzyme directly, rather than a lack of free dolichol-PO₄. These studies link conA resistance, cell surface glycoprotein alterations, and defective mannosyl transferase activity with the inability to carry out normal cellular differentiation to form multinucleated myotubes.     

The fluorescence scanning of microgels stained for glycoproteins with fluorescein isothiocyanate-labelled concanavalin A.

A technique is presented which allows one to label and quantitate glycoproteins. Small amounts of protein from biological samples (0.5-2.5 microgram for mixtures and less for individual proteins) are separated by sodium dodecyl sulfate gel electrophoresis on 1-30% polyacrylamide gradient microgels. The gels are stained with Co-omassie Brillant Blue R250 to evaluate relative migration or fixed in 2-propanol/acetic acid and stained with fluorescein isothiocyanate-labelled concanavalin A. The microgels are then scanned using a fluorescence microscope controlled by a computer, although simpler configurations are possible. Standards of known carbohydrate composition (e.g., glucose oxidase) are used for comparative purposes. Glycoproteins in the order of 5-30 ng protein (or 1-5 ng carbohydrate) can be detected without difficulty. This technique may prove valuable in evaluating glycoproteins when the available material is limited.