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A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor—iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125 I- and 32 P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [ 125 I]insulin and [ 125 I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 μg of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125 I-labelled ligand—soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

The antigens of rat embryo fibroblasts (REF) and of rat Rous sarcoma cells (derived by in vivo passage of oncogenically transformed REF) were studied using the technique of non-ionic detergent solubilization of radiolabelled cells. Solubilized antigens were complexed with rat immune IgG, and precipitation of the complexes was accomplished with rabbit anti-rat IgG. The precipitated radiolabelled antigens were then dissolved in sodium dodecyl sulfate and separated by poly-acrylamide gel electrophoresis. This investigation disclosed the existence of cell surface antigenic proteins which are derived from the fetal calf serum (FCS) used in the cell-culture medium. These FCS-dependent antigens include at least three molecular species of approximate molecular weights 95,000, 80,000 and 68,000 daltons. They are probably derived from simple adsorption of FCS proteins to the cell surface, although more complex interactions are possible. One of these proteins (95,000 daltons) is of particular interest. It tenaciously adheres to the cell surface so that a trace amount remains even after subculture in the absence of FCS. Rat Rous sarcomas which are morphologically highly transformed appear to bind very little or none of this protein to their surfaces, whereas untransformed rat embryo fibroblasts bind large quantities. A rat Rous sarcoma line which is intermediate in morphological transformation binds an intermediate amount of this antigen. These findings invite speculation that the interaction of certain serum components with the cell surface may be related to plasma membrane properties which distinguish untransformed and transformed cells.

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.

Glucose deprivation of chick embryo fibroblasts results in a cycloheximide-sensitive stimulation of hexose transport and an increase in the levels of glucose-regulated polypeptides of molecular weights 75 000 and 95 000. The relationship between these two phenomena is evaluated in this study. The glucose deprivation-induced stimulation of hexose transport was observed to occur in two phases: a rapid (complete by 15 min) cycloheximide-insensitive increase of 50-100% and a slower (observable by 6 h) cycloheximide-sensitive increase in transport to about five times the basal level. The time course of the latter increase preceded that of the appearance of the 75 000 and 95 000 dalton polypeptides; by the time that increases in the levels of these polypeptides were observed, the hexose uptake rates had almost reached their maximum value. Upon cellular fractionation, the greatest enrichment of the 75 000 and 95 000 dalton polypeptides was observed in the endoplasmic reticulum fraction, which was devoid of vesicular stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity but not in the 75 000 and 95 000 dalton polypeptides. The glucose deprivation-induced increase in hexose uptake was not prevented by tunicamycin, although this inhibitor of protein glycosylation decreased the hexose uptake of glucose-fed cells by 80% after 24 h. However, under these latter conditions an increase in the levels of the 75 000 and 95 000 dalton polypeptides was observed. On the basis of this data, we conclude that the polypeptides of molecular weights 75 000 and 95 000 are not involved in glucose transport.

Uptake of 2-deoxy-d-glucose in cultured chick embryo fibroblasts consists of two reactions, transport and phosphorylation (Kletzien, R. F., and Perdue, J. F. (1973) J. Biol. Chem. 248, 711–719). Further studies were initiated to examine in greater detail the mechanism of sugar uptake and to determine which of the two reactions was responsible for the differing rates of 2-deoxy-d-glucose uptake between rapidly and slowly growing cells. The uptake of 2-deoxy-d-glucose was studied as a function of time and substrate concentration to establish the conditions under which the phosphorylation of the sugar parallels uptake. The initial rates of phosphorylation of 2-deoxy-d-glucose in intact cells were dependent on the rate of cell growth. However, the initial rates of phosphorylation in cellular homogenates prepared from rapidly and slowly growing cells were identical and were 4 to 6 times greater than the rates observed in intact cells. Experiments with cytochalasin B, a sugar transport inhibitor, demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation. These data indicate that a reaction prior to phosphorylation is responsible for the altered rates of sugar uptake in rapidly and slowly growing cells and that this same reaction limits the rate at which 2-deoxyglucose is phosphorylated in intact cells at early time points. The kinetic constants for the uptake and phosphorylation of 2-deoxy-d-glucose and the inhibition of this uptake and phosphorylation by competing sugars were calculated from Lineweaver-Burk plots. The Km for sugar uptake was constant at 2 mm; the Vmax was 16.7 or 33.2 nmoles per mg of protein per min, depending on the rate of cell growth. The Km for sugar phosphorylation in cellular homogenates was 0.8 mm; the Vmax was 75 nmoles per mg of protein per min and was not dependent on the rate of cell growth. The Ki values for the competitive inhibition of 2-deoxy-d-glucose uptake in intact cells and phosphorylation in cellular homogenates by d-glucose were 2 and 0.18 mm, respectively. 3-O-Methyl-d-glucose competitively inhibited 2-deoxy-d-glucose uptake with a Ki of 17 to 20 mm. The kinetic constants for 3-O-methyl-d-glucose uptake and inhibition of uptake by d-glucose were also calculated from Lineweaver-Burk plots. The Km for 3-O-methyl-glucose uptake was 3.5 to 5.0 mm, and the Vmax was 19 or 37.5 nmoles per mg of protein per min, depending on the rate of cell growth. The Ki for d-glucose inhibition of uptake was 1.8 mm. These results demonstrate that 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-glucose are all transported by the same site, that sugar transport is the rate-limiting step in sugar uptake, and that sugar transport is a reaction distinct from phosphorylation. The latter conclusion was further substantiated by the finding that hexokinase was not associated with isolated plasma membrane. The difference in the rate of uptake of 2-deoxy-d-glucose and 3-O-methyl-d-glucose between rapidly growing and slowly growing cells was strictly attributed to differences in the Vmax for sugar transport; the Km for transport did not change with the rate of cell growth. Activation energies for the sugar transport reaction were calculated from Arrhenius plots and were found to be identical for rapidly and slowly growing cells, a result which is consistent with the interpretation that the increase in Vmax in rapidly growing cells is the result of a greater number of sugar transport sites.

The mode of induction of sugar transport by serum-stimulation of growth and hexose-starvation in chick embryo fibroblasts (CEF) has been studied using metabolic inhibitors. We have concluded from these studies that the sugar transport increases induced by serum-stimulation are regulated by post-transcriptional mechanisms while sugar transport increases induced by hexose-starvation are regulated by a transcriptional mechanism. CEF infected with a temperature-sensitive mutant of the Rous sarcoma virus. Ts68 and incubated at the nonpermissive temperature for transformation, 41 degrees, retain the capacity to regulate sugar transport in a manner similar to uninfected CEF. However, Ts68-infected CEF maintained at the permissive temperature for transformation, 37 degrees, have lost the ability to regulate sugar transport at the post-transcriptional and post-translational levels.

A large molecular weight polyaldehyde fixative (macrofixative), prepared by periodate oxidation of the branched polysaccharide dextran, was used for stabilizing the cell surface membrane. Evidence indicated that this substance is non-toxic to cultured cells and that its action is probably restricted to external surfaces. Macrofixation of chick embryo fibroblasts (CEF) was found to inhibit cell growth, but it did not interfere with basal cellular metabolic processes as assessed by the ability to transport and phosphorylate sugar. Cultured chicken cells which have been treated with macrofixative could be lysed in a hypotonic medium without detachment of the evacuated plasma membrane from the substratum. In situ cell ghosts, prepared in this manner, provided the following immunoelectron microscopic findings: ( a ) lysed cells could be used as electron-translucent specimens for two-dimensional ultrastructural analysis of ferritin-labeled antigen-antibody complexes; ( b ) cell surface antigens of previously lysed cells could be redistributed into small patches by reaction with specific antibody, but more complex redistribution patterns (such as cap-like foci of coalescence) did not occur; ( c ) thin sections of lysed, virus-infected CEF, in which cell surface-associated viral antigens were labeled with ultrastructurally visible markers, revealed that the determinants are expressed only on the external side of the plasma membrane. Treatment of living virus-infected cells with macrofixative as an external cross-linking reagent did not significantly alter any patterns of antibody-induced redistribution of cell surface viral antigens.

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.

1. 1. Plasma membrane was isolated from cultures of chick embryo fibroblasts which were converted by avian sarcoma viruses, morphf Fujinami virus, and a mutant of this virus, morphr Fujinami virus. The morphf virus-converted cells were fusiform in shape, whereas fibroblasts converted by the morphr virus became round. 2. 2. Cell membrane isolated from the morphr virus-converted fibroblasts had an increase of more than 40 % in neutral sugar content compared with that isolated from uninfected cells or cells converted by morphf Fujinami virus. 3. 3. The sialic acid content of the cell membrane isolated from cells converted by oncogenic viruses was decreased compared with that of plasma membrane from RAV-49-infected cells or uninfected cells. Since this decrease was found in cells converted by sarcoma-producing viruses but not in those infected with a leukemia virus, and since the decrease was not correlated with cell shape, it represents a fundamental alteration in membrane chemistry paralleling a loss of the fibroblasts' controls of growth and multiplication.

Secretory granules from anterior pituitary glands of young adult male castrate rats were isolated by differential centrifugation, microfiltration, and discontinuous density gradient centrifugation. The granules were obtained as pellets, sectioned, and studied with the electron microscope. A major part of the gonadotropin and a substantial amount of the TSH were associated with the isolated granules. Negligible amounts of growth hormone and prolactin were present as contaminants. Succinic dehydrogenase, glucose-6-phosphatase, acid protease, and acid and alkaline phosphatases were not found in the granules. Alkaline protease was the only enzyme found to be associated with the granules, and it is suggested, in the light of these results, that the alkaline protease may be involved in the release of the hormones.

A mold metabolite, cytochalasin B, inhibits uptake of 2-deoxy-d-glucose in chick fibroblasts and cultured liver cells. This inhibition is rapid, reversible, and is achieved at very low concentrations of the metabolite. Cytochalasin A also inhibits sugar uptake, but the magnitude and reversibility of inhibition differs from that produced by cytochalasin B.

A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.

Competitive binding techniques were used to study the interaction of insulin-like growth factor I (IGF-I) with a plasma membrane-enriched subcellular fraction purified from primary breast and colon carcinoma specimens obtained at surgery. The presence of specific binding sites for IGF-I was detected in all tumour specimens studied. Scatchard analysis and competition studies with insulin and insulin-like growth factor-II (IGF-II) revealed the presence of specific IGF-I receptors, showing a Kd-value of approximately 2 nM. These results are consistent with the hypothesis that somatomedins play a role in determining the proliferative behaviour of human breast and colon tumors, and suggest that recent laboratory studies showing dependence of neoplastic cells on somatomedins for optimum proliferation may have clinical relevance.

Human serum contains two classes of somatomedins (Sin) with similar three-dimensional structures and mass (i.e., ~7500 kdaltons (kd)), but that can be distinguished from each other by their isoelectric point (pI) values. Those with alkaline pI's include the presumably identical molecules of insulin-like growth factor I (IGF-I), SmA, SmC, and basic Sm. In humans, the neutral–acidic class of Sm is represented by IGF-II. The sera of rats and other animals contain similar classes of Sm. In normal children and adults, plasma levels of both IGF-I and -II are under growth hormone control, although only elevated concentrations of the former can be positively correlated with adolescent skeletal growth. Elevated blood levels of IGF-II or an embryonic form of it, however, are correlated with fetal development of rats, sheep, and possibly in humans. As evidenced from competitive radioreceptor binding studies and affinity-labelling techniques, receptors that bind insulin and IGF-I are immunologically, structurally, and functionally related: (a) monoclonal and polyclonal antibodies to insulin receptors recognize determinants on receptors that bind IGF-I; (b) both receptors have 140-, 95-, and possibly 45-kd subunits that are interchain disulfide-bonded to form glycoprotein complexes of relative masses (Mr) 300 000 – 400 000; and (c) occupancy of the 140-kd subunits by hormone or growth factor stimulates the phosphorylation of tyrosine residues on the 95-kd subunit by receptor-associated protein kinases. Receptors that bind IGF-II are very different from insulin or IGF-I receptors: (a) there is no evidence of immunological cross-reactivity to the insulin receptors or of intrinsic protein kinase activity and (b) estimates of the molecular weight of the detergent-solubilized IGF-II receptor from its hydrodynamic properties or from its electrophoretic mobility during quantitative or sodium dodecyl sulfate–polyacrylamide gel electrophoresis established it to be a single-chained glycoprotein of MT 220 000 held together by intrachain disulfide bonds.

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.

Sarcomas were initiated in chicken muscle and wing web by Bratislava 77 and morph r Fujinami virus. Plasma membrane was isolated from the virus-induced tumor cells by differential centrifugation and flotation equilibrium centrifugation. The levels of neutral sugar and sialic acid in these isolated plasma membranes were very similar to the levels found in cultured chick embryo fibroblasts transformed in vitro with the same oncogenic viruses and differed markedly from the levels found in uninfected and leukosis virus-infected fibroblasts. The phospholipid content of the isolated cell membranes from tumors was less than the quantity of lipid found in the plasma membrane of cultured cells and differed with the site of the tumor. Breast muscle tumors contained less plasma membrane phospholipid than did wing tumors. The similarities in the neutral sugar and the sialic acid content of these two different sources of plasma membrane indicate that oncogenic transformation in cell culture reproduces in situ neoplastic change to a large extent, for at least this one parameter of cell surface change.

1. 1. Plasma membrane was isolated from cultures of chick embryo fibroblasts and fibroblasts which had been infected with the avian sarcoma virus, RBA, and the avian leukosis virus, RAV-49. 2. 2. On continuous density gradients of sucrose, the cell membrane was localized in two fractions, the A′ band and B′ band. The membranes in the A′ band contained twice as much phospholipid and cholesterol as the membranes in the B′ band. The membranes in the latter band also contained less neutral sugar and sialic acid. 3. 3. Plasma membrane in the A′ band that was isolated from the oncogenic virus-converted cells, RBA, had increased levels of neutral sugar (358 vs. 243 μg per mg protein), but the sialic acid content of this membrane was decreased by 25 %. 4. 4. Cell membrane obtained from the leukosis virus-infected cells had quantities of sialic acid and neutral sugar that were similar to those of uninfected cells. Therefore, the increase in neutral sugar and the decrease in sialic acid found in cell membrane from the RBA-converted cells are chemical modifications of the cell surface which reflect conversion by an oncogenic virus and are not the result of virus infection per se.

1. 1. Cell membrane was isolated by flotation equilibrium centrifugation of a homogenate from cultured epithelial cells derived from rat liver, BRL cells, and from BRL cells which had been converted by Murine Sarcoma Virus (MSV). 2. 2. On continuous density gradients of sucrose, the plasma membrane was localized in two fractions, the A′ band and the B′ band. Phospholipid, cholesterol, sialic acid, neutral and amino sugar, CTPase (EC 3.6.1.4), and AMPase (EC 3.1.3.5) were concentrated in the isolated membranes from the BRL cells. 3. 3. Plasma membrane from the MSV-converted BRL cells was similar to that of the BRL cells in chemical composition but was markedly deficient in nucleotide di- and triphosphatases. AMPase, which was very high in the BRL cells, was increased still further in the virus-converted cells.