Most iodinatable fibroblast surface proteins accompany the cytoplast membrane during cytochalasin B-mediated enucleation of chick embryo fibroblasts.

Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments.

Transformation-sensitive isoactin in passaged chick embryo fibroblasts transformed by Rous sarcoma virus.

Transformation by Rous sarcoma virus (RSV) has been reported to block the expression of differentiated cell products in chicken cells. The expression of these proteins may or may not be suppressed when temperature-sensitive mutants are shifted from the nonpermissive to the permissive temperature. A general characteristic of cellular transformation is the disruption of the microfilament system. In passaged chick embryo fibroblast cultures (CEF), this system is principally composed of isomeric forms of actin designated alpha, beta, and gamma by their isoelectric focusing and when subjected to SDS-PAGE behavior. We present evidence that an alpha-actin in CEF cultures, identified by its electrofocusing behavior, retention in the cytoskeleton, and DNase 1 binding properties, is selectively and dramatically reduced in amount upon transformation by RSV. Little or no reduction is observed in the beta- and gamma-isoactins. The reduction of alpha-actin is shown to be reversible and transformation related by use of a temperature-sensitive mutant, tsNY68. The decrease in this transformation-sensitive isoactin is apparently due to a decrease in synthesis, though other possibilities are discussed. A specific decrease in a particular isoactin after transformation may give insight into the mechanism by which the microfilaments are normally maintained.

Differential binding of chemokines to glycosaminoglycan subpopulations.

BACKGROUND: Specificity in leukocyte trafficking is likely to depend on sequential interactions between various cell-type-specific leukocyte adhesion molecules, such as selectins and integrin ligands, and leukocyte-activating factors. A major class of leukocyte-activating factors, the chemokines, are soluble polypeptides that bind glycosaminoglycans, the polysaccharide components of cell-surface and extracellular-matrix proteoglycans. It has been suggested that cell-surface glycosaminoglycans of the heparin/heparan sulfate class mediate the presentation of chemokines to leukocytes by vascular endothelial cells. We investigated the possibility that specificity exists in the recognition of particular heparin/heparan sulfate structures by chemokines, by studying the binding of four members of the chemokine superfamily to heparin and heparan sulfate. RESULTS: Using affinity co-electrophoresis we found that interleukin-8 preferentially bound a subfraction of heparin that also showed increased affinity for melanoma growth stimulating activity (also known as MGSA, GRO or GRO alpha). This same subfraction of heparin, however, was not significantly preferentially bound by platelet factor 4 or neutrophil activating factor-2. Subsequent analysis of the three-dimensional structures of these chemokines indicated that their ability to discriminate among heparin subspecies correlates with the presence of paired glutamic acid residues within the putative glycosaminoglycan-binding site of the chemokine. This observation led to predictions about the relative affinities of heparan sulfate for interleukin-8 and platelet factor 4, predictions that were confirmed by further binding assays. CONCLUSION: Chemokines can bind selectively to subsets of heparin/heparan sulfate glycosaminoglycans, raising the possibility that glycosaminoglycans participate in determining the specificity of leukocyte recruitment in vivo.

Identification of the transferrin receptor of the rabbit reticulocyte.

Reticulocytes were separated on the basis of density by isopycnic centrifugation in dextran gradients. This parameter was shown to correlate with the degree of maturity of the cells. Lactoperoxidase iodination of cells of different densities followed by sodium dodecyl sulfate (NaDodSO4) electrophoresis revealed a 190 000 molecular weight protein which was well labeled in early reticulocyte membranes. Efficiency of labeling decreased as the cells increased in density, and high specific activity iodination of mature erythrocytes did not result in the labeling of any species near this molecular weight. Inclusion of rabbit transferrin prior to the iodination procedure resulted in a specific loss of labeling of this 190 000 molecular weight species. When steps were taken to clear endogenous transferrin from the membranes, the labeling of this species was enhanced. These observations are consistent with the concept that transferrin can block the lactoperoxidase catalyzed iodination of this membrane protein by specifically associating with it. Coomassie blue and periodic acid-Schiff staining of NaDodSO4 gels of these membranes revealed that a glycoprotein present at this molecular weight is lost during the course of reticulocyte maturation. It is concluded from these studies that a glycoprotein of molecular weight 190 000 constitutes the transferrin receptor in the reticulocyte membrane.

Specific dephosphorylation of membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts (CEF) infected with avian sarcoma virus become rapidly transformed as a result of expression of the viral src gene in the form of a single polypeptide of molecular weight 60,000 (pp60src) with protein kinase activity and suggested preferential association with the plasma membrane. Studies with normal avian and mammalian cells have revealed the presence of an antigenically related protein which seems to have similar kinase activity, but which is present at less than 1% of the levels of virally induced src protein found in transformed cells. As dynamic phosphorylation is important in numerous regulatory processes, the phenotypic expression of transformation may arise from an imbalance in one or more regulatory mechanisms that are controlled by protein phosphorylation. The cell membrane is affected during transformation, including its phosphotransferase activity. The latter has been shown using isolated membrane fractions whose properties may be changed during preparation. Therefore, we have compared the phosphorylation state of individual membrane proteins found in intact normal and RSV-transformed cells and report here the identification of two heavily phosphorylated, acidic membrane proteins in normal CEF which are specifically dephosphorylated on transformation by wild-type and temperature-sensitive Rous sarcoma viruses.

An alternative model for the binding and release of diferric transferrin by reticulocytes.

The biphasic binding of diferric transferrin to reticulocytes has been reevaluated with a series of kinetic and equilibrium studies. Identical binding progress profiles were observed for reticulocytes in the presence or absence of oxygen. The relative size of the rapid initial adsorption step could be increased to ca. 65% of the total binding by stripping the cells of endogenous transferrin or reduced to 0% by preloading the cells with nonradiolabeled diferric transferrin. Preloading the cells with 125I-labeled diferric transferrin and chasing with 131I-labeled diferric transferrin revealed identical rate constants for release and binding. Scatchard plots of equilibrium binding of diferric transferrin to reticulocytes showed no significant effects of anaerobiasis or 2,4-dinitrophenol on the equilibrium binding constant or the maximum number of binding sites. The potent microtubule inhibitor nocodazole had no effect on the progress curves for transferrin binding or iron uptake by reticulocytes. It was concluded that the rapid adsorption step in the binding profile represents binding to open receptors and that the slow first-order binding phase represents binding of radiolabeled transferrin to receptors already occupied by nonlabeled endogenous transferrin as this endogenous transferrin leaves the receptors. Furthermore, this first-order binding phase, unlike iron uptake, does not require the presence of active oxidative phosphorylation. These findings are consistent with a specific desorption-adsorption model for the interaction of diferric transferrin with reticulocytes.

An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay.

A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.