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The analysis of cDNA clones for human (1,2) and rat (3) insulin-like growth factor-II (IGF-II)1 has led to the prediction that the processed forms of the growth factors, i.e. Mr = 7422 (67 amino acids) for human IGF-II are synthesized as precursors with an extension of 89 amino acids at the carboxyl terminus. This extension is termed the E domain. Moses et al (4) identified two precursor forms of rat IGF-II (originally designated multiplication-stimulating activity) in the conditioned medium of Buffalo rat liver, i.e. BRL-3A, cells with appMrs = 16,270 (MSA-1) and 8,700 (MSA-II). Human serum, spinal fluid and tissue extracts also contain high Mr forms of IGF-II (5-8). Zumstein et al., (5) have purified a Mr = 10,000 variant form of IGF-II from serum that contained Cys-Gly-Asp for Ser33 in the C domain and an E domain extension of 21- amino acids. This 10 kDa IGF-II may be similar or identical to the “big IGF-II” that was reported to be present in human serum and in spinal fluid (6). We have isolated a still larger form of IGF-II from normal human serum (7). N-terminal amino acid sequence analysis through the first 28 residues and RRAs using rat placental membranes established it as a form of IGF-II. As evidenced from its mobility during SDS-PAGE, it has an appMr = 15,000. Very recently, Hudgins et al., (8) established that normal human serum contains several forms of precursor IGF- II with acidic isoelectric points, i.e. pI’s. The mass and acidic nature of one of these molecules with an apparent Mr= 15,000 was contributed, in part, by polysaccharides and sialic acids, respectively (8). These results may explain the observations of several investigators that extracts from the tissues and serum of patients with malignant tumors contain a broad size range of IGF-II (2,9,10).

Chick embryo fibroblasts, infected with a temperature-sensitive mutant of the Rous Sarcoma virus Ts-68 and cultured at the permissive temperature (37°C) for transformation, exhibit an increased V max for sugar transport, compared to that of uninfected fibroblasts and infected fibroblasts cultured at the nonpermissive temperature (41°C) for transformation. Studies were initiated to determine which events in the regulation of transport could account for these differences. Three experimental models were used which result in an increase in the V max for sugar transport: growing the cells in the absence of glucose; shifting the virus-infected cells from 41°C to 37°C; and adding fetal calf serum to serum-deprived cultures. Culturing Ts-68-infected cells at 41°C or 37°C in glucose-free medium for 30 hr resulted in a similar induction of the synthesis of the sugar transport system. This result established that the synthesis of the transport system was not temperature-sensitive in infected cells maintained at 41°C, nor was there a difference in the rate of its degradation. The half-life (t12) of the transport system for cells grown at 37°C was about 5.2 to 6 hr, and was the same for both virus-transformed and uninfected fibroblasts. The t12 for cells cultured at 41°C was also similar for uninfected and infected cells, and was between 3.1 and 3.4 hr. Experiments using cordycepin demonstrated that a metabolic step blocked by this inhibitor was required for the enhancement of sugar transport in Ts-68-infected fibroblasts following a shift in culture temperature from 41°C to 37°C. Actinomycin D was without effect. Ts-68-infected cells cultured at 41°C and uninfected cells both exhibited a rapid decay of transport activity when fetal calf serum was removed from the medium and a biphasic increase in activity following its addition. The rate of sugar transport in Ts-68-infected cells cultured at 37°C was not affected by the removal or addition of serum, indicating that one aspect of transformation is the loss in the ability of cells to effect post-transcriptional and post-translational regulation of the number of sugar transport sites.

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of complexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the “capping” phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37° C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites – perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.

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.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.

The removal of serum from the medium of uninfected fibroblasts decreased the rate of uptake of uridine, 2-deoxyglucose, alpha-aminoisobutyrate and thymidine. Its subsequent addition rapidly and reversibly stimulated the uptake of all the nutrients but thymidine and this response was not inhibited by treatment of the cells with cycloheximide. The cycloheximide insensitive, rapid increase in the rate of transport has been designated post-translational control. The nutrient transport systems in chick embryo fibroblasts transformed in vitro with avian sarcoma viruses and virus-induced cultured chicken tumor cells do not respond to serum removal or addition. Two possible levels for the control of nutrient transport, i.e., mitogen receptor occupancy and mitrogen-induced activation of the transport system, are presented to explain these observations.

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.

Plasma membranes, isolated from uninfected cultures of chick embryo fibroblasts, were dissociated with sodium dodecyl sulfate and analyzed for glycoprotein and protein subunit composition by discontinuous gel electrophoresis. More than 30 protein subunits and 18 glycoprotein subunits were obtained from the A′ and B′ plasma membrane fractions; the membranes in the A′ fraction contain almost twice as much phospholipid and cholesterol as those of the B′ fraction and have a density of 1.06 versus 1.12. Electrophoretic separation profiles were similar for both types of membranes. The molecular weights of the components range from more than 220,000 to less than 22,500. Major protein subunits had molecular weights of 175,000, 95,000 75,000, 69,000, 56,000, 47,000 35,000, and 32,000. The majority of the glycoprotein subunits migrated with protein subunits of more than 70,000 molecular weight. No estimation of molecular weights was attempted for these components. Iodination of whole cells yielded A′ and B′ membrane fractions, which were differentially labeled, with five and seven peaks of radioactivity, respectively. Some degree of differentiation between A′ and B′ fractions was also obtained when cells were prelabeled with radioactive glucosamine and mannosamine. A′ membrane fractions, known to contain more lipid than B′ membrane, also showed a greater recovery of isotope in the lipid region of the gel than did B′ membrane.

Membranes have been isolated from fresh rectal glands of the dogfish shark, Squalus acanthias, in which the specific activity of the (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) is as high as 400 µmoles of Pi per mg of protein per hour. Membranes isolated from frozen rectal glands have only one-fourth to one-third this specific activity. Solubilization of membranes from frozen glands with the nonionic detergent Lubrol WX activates the NaK ATPase 2-fold, but there is no activation of the enzyme with membranes from fresh glands. Purification of Lubrol extracts by zonal centrifugation and a novel ammonium sulfate fractionation gives an enzyme preparation with a specific activity of 1,500 µmoles of Pi per mg of protein per hour. The same specific activity and gel pattern is obtained with enzyme purified from membranes from either fresh or frozen glands. The loss of Lubrol activation of the enzyme from frozen glands occurs on zonal centrifugation, where free Lubrol is removed. The over-all yield of enzyme from the membrane stage is 70%. A mince of 10 rectal glands weighing about 10 to 15 g fresh weight yields about 20 to 30 mg of purified enzyme. The enzyme is completely stable at 0° for 10 days in either the membranous form or in the most highly purified form. It is stable on freezing at -70°. Disc gel electrophoresis shows a gradual elimination of proprotein bands on purification. At the final stage of purification, scanning of Coomassie blue-stained gels gives 72% of the protein as the 97,000 molecular weight catalytic subunit, 19% as the 55,000 molecular weight glycoprotein, and 8.5% as the protein running with the tracking dye. The catalytic subunit and the glycoprotein can be isolated in milligram quantities by solubilization and chromatography on Sephadex G-150. About 90% of the protein applied to the column can be recovered. Sephadex chromatography gives a protein composition of 66% for the catalytic subunit, 28% for the glycoprotein, and 5% for the protein running with the tracking dye. The catalytic subunit and the glycoprotein enrich more or less in parallel as purification proceeds. At the last stage of purification, both proteins are found in the form of vesicles, which bear a striking resemblance on negative staining to purified cytochrome oxidase. Electron-translucent rods and rings which are approximately 80 A in diameter are also formed with projections of about 35 to 55 A at regular intervals. These rods and rings resemble reconstituted oligomycin-sensitive mitochondrial ATPase. The projections appear to be more hydrophilic, and this, coupled with their size, suggests that they are the glycoprotein. Incubation of the enzyme with [γ-32P]ATP, magnesium, and sodium gives 4,080 pmoles of acyl phosphate per mg of protein, which is 2 to 3 times higher than the highest levels of phosphorylation previously reported, thus attesting to the high purity of the enzyme preparation (the level of phosphorylation is generally accepted as being proportional to the number of NaK ATPase molecules in the preparation). The turnover number of the preparation is 6,300 min-1, which is within the range found for most NaK ATPase preparations from different organs and species. Arguments are presented for and against the view that the glycoprotein is a subunit of the NaK ATPase. If the catalytic subunit of molecular weight of 97,000 and the glycoprotein of molecular weight of 55,000 are both integral components of the NaK ATPase, the enzyme is 90 to 95% pure. If the 97,000 molecular weight subunit is the only subunit in the enzyme, the present preparation is 66 to 72% pure.

Cultured chick embryo fibroblasts accumulate Ca2+ in the presence of Mg2+ and ATP. The uptake is highly specific; Mn2+ inhibits it, and other nucleotide triphosphates are without effect. The presence of oxalate, Na+ and/or K+ increases the amount of accumulated Ca2+. The cation transport is inhibited by mersalate, oligomycin, and hydroxylamine. The capacity of fibroblasts to energetically transport and/or bind Ca2+ resides in components of the plasma membrane. The function of this Ca2+ uptake may be to control motility in ameboid-like cells.

The outer membrane of beef heart mitochondria is composed of a detachable sector (S) and an invariant sector (K). These separate fractions (K and S) have been isolated and examined in detail, and the results were described in the preceding two communications. This communication describes the preparation and properties of the composite outer membrane fraction. This membrane fraction contains all the enzymes of the citric acid cycle and other ancillary enzyme systems present in the S and K fractions. As isolated, it is essentially devoid of any electron transfer components, accounts for the estimated mass of the outer membrane, contains about 25–30% phospholipid by dry weight, and resembles the intact outer membrane.

The mold metabolites, cytochalasin A and B, have been shown to be potent inhibitors of 2-deoxy-d-glucose uptake in cultured cells (Kletzien, R. F., Perdue, J. F., and Springer, A. (1972) J. Biol. Chem. 247, 2964–2966). Cytochalasin B also inhibits the uptake of d-galactose and d-glucosamine in chick embryo fibroblasts at concentrations similar to those used to inhibit the uptake of 2-deoxy-d-glucose. A concentration of cytochalasin B which inhibited 50% of sugar uptake did not affect the ability of the cells to concentrate 2-deoxy-d-glucose as the phosphorylated sugar nor did the drug inhibit the phosphorylation of 2-deoxy-d-glucose by cellular homogenates. Therefore, the inhibition of sugar uptake by cytochalasin B was due to an inhibition of transport across the plasma membrane. This inhibition was analyzed by three methods and was of the linear competitive type with an apparent dissociation constant (Ki) for the inhibitor-carrier complex of 1.45 x 10-7 m.

The (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) in Lubrol extracts of NaI-treated bovine brain microsomes has been purified 30 to 50 times over that in the microsomes by salt and isoelectric precipitation, zonal centrifugation, and a new ammonium sulfate fractionation procedure. The final step in the purification procedure renders the NaK ATPase insoluble. The partially purified enzyme has been examined by polyacrylamide gel electrophoresis after solubilization in either phenol-acetic acid-urea or sodium dodecyl sulfate-mercaptoethanol. The former system is unsatisfactory in that a considerable proportion of the protein, including the NaK ATPase, remains at the origin; however, all of the protein penetrates the gel with the latter system. The purification procedure removes the majority of proteins seen in the microsomal fraction. With the partially purified enzyme, one prominent band, two less prominent bands, and two to three rather minor bands are seen with the sodium dodecyl sulfate system. The most prominent band has been identified as the phosphorylated subunit of the NaK ATPase by labeling its glutamyl-γ-phosphate residue with 32P by prior incubation of the partially purified enzyme with ATP-γ-32P in the presence of magnesium and sodium. Its apparent molecular weight is 94,000. Scanning of the protein-stained gel indicates that 25 to 40% of the protein applied to the gel can be accounted for by this 94,000 molecular weight subunit. The protein, phospholipid, and carbohydrate content of the preparation remain remarkably constant as purification proceeds, being approximately 50%, 25%, and 2 to 3%, respectively, of the dry weights of the preparations after correction for bound Lubrol. The protein-bound Lubrol at the final stage of purification is 17%. The cholesterol content falls with purification. Ninety-nine per cent of the ATPase in the partially purified enzyme is ouabain-sensitive. Ouabain-sensitive, potassium-activated p-nitrophenylphosphatase activity parallels NaK ATPase. All detectable cholinesterase in the microsomes is removed by purification. With the method of purification reported here, as much as 1.2 kg of bovine brain cortex can be worked up weekly giving 30 to 50 mg of final enzyme with a specific activity of 450 to 750 µmoles of ATP split per mg of protein per hour. Based on the gel scans and on calculations from the picomoles of phosphorylated subunit and molecular weight of the native enzyme, the best preparations are judged to be approximately half pure. The partially purified enzyme is quite stable on storage at 0°, retaining full activity for two months. Electron microscopy of the enzyme preparation after zonal centrifugation shows rods of about 60 A in diameter, spherical protein, and aggregated protein. The ammonium sulfate fraction shows chains of protein averaging 60 A in diameter, which are believed to be end-to-end aggregates of the rods seen in the zonal fraction. Many chains form ringlike structures. Sheets of aggregated protein are also seen. Resolubilization of the ammonium sulfate enzyme with Lubrol to protein ratios of 20:1 shows predominantly protein subunits averaging 60 A in diameter and some aggregates of subunits.

A mitochondrial preparation containing the complete electron transfer chain (EP 1 ) as well as a preparation containing only one sector of the chain (Complex IV) were resolved into structural and catalytic protein fractions. The respective oxidation-reduction components of EP 1 and Complex IV, as well as the lipid, were concentrated exclusively in the catalytic protein fraction. Integrated electron transfer activity appears to be unaffected by the removal of structural protein. Such losses in electron transfer activity as are sustained appear to be referable to the severity of the procedure for resolution rather than to the removal of structural protein. Under appropriate conditions structural protein will recombine with catalytic protein to “reconstitute” a particle that resembles the parent particle in respect to solubility, activity, spectral properties, and ultrastructure. The membrane-forming capability of the complexes is not affected by the removal of structural protein.

A method is described, involving digestion of mitochondria with phospholipase, which leads to a clean separation of the two mitochondrial membrane systems. An inner membrane fraction, R 2 , can be prepared which is essentially free from the activities known to be associated with the outer membrane, but is enriched in components of the inner membrane and in some of its main activities (electron transfer, and ATPases stimulated by divalent cations). Electron microscopy shows that the R 2 fraction is an essentially complete, unruptured inner membrane. Ca ++ ions translocated into the mitochondrion are completely recovered in the R 2 fraction; this has been taken as support for the premise that the R 2 particle is a morphologically intact inner membrane. The fact that sequences which depend upon energy transformations (oxidative phosphorylation and energized translocation) are partially lost in the inner membrane fraction, R 2 , suggests that, although morphologically intact, the functionality of the inner membrane has been somewhat damaged by the digestive procedure.

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.

An actin-like protein was isolated from the acetone powder of glycerinated chick embryo fibroblasts and purified to electrophoretic homogeneity as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified protein has a molecular weight of 45,500, resembles rabbit actin in electrophoretic mobility and ultrastructure, undergoes reversible globular-fibrous transformations, and combines with rabbit myosin to form hybrid actomyosin. This actomyosin exhibits ATPase activity and superprecipitation properties similar to those of actomyosin from rabbit muscle. In fibroblasts, the actin-like protein may function in the maintenance of shape and in the implementation of intra- and extracellular movements.

The properties and distribution of nucleotide phosphohydrolase were examined in intact and broken preparations of chick embryo fibroblasts. These cells have a specific ADPase (EC 3.6.1.6) associated preferentially with the plasma membrane. This nucleotide diphosphate was hydrolyzed at an optimum of pH 7.8 and at a Mg 2+ to ADP ratio of 1:1 . The conditions for maximum hydrolysis of ATP by ATPase (EC 3.6.1.3) were established with respect to protein concentration, pH and mono- and divalent ion specificity and concentration.

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.

Triton X-100 or Nonidet P40-deoxycholate extracts of [3H]fucose-labeled Rous sarcoma virus-transformed chick embryo fibroblasts were examined by indirect immunoprecipitation for the presence of a tumor-specific neoantigen of 100,000 daltons. Extracts were incubated with immune IgG from Rous tumor-sensitized chickens, and the resultant antigen-antibody complexes were precipitated with rabbit antichicken IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactivity appeared between the migration positions of proteins having molecular weights of 65,000 and 95,000 daltons and at about 30,000 daltons. These antigens were group-specific, and thei precipitation could be inhibited by competition with extracts from cultured fibroblasts that had been infected with a nontransforming avian leukosis virus. They were not precipitated with IgG from unimmunized chickens or chickens immunized with the culture supernatants of uninfected chick embryo fibroblasts. In contrast to results reported recently, the present results could not confirm by immunoprecipitation, the existence of a tumor neoantigen different from that associated with viral components. However, the tumor-specific antigen possibly existed on the cell surface but was not preserved in these detergent extracts.

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.

Electron microscopic evidence is presented for membrane formation by the repeating units of (1) the individual complexes of the electron transfer chain; (2) the outer membrane of beef heart mitochondria; (3) the mitochondrial membranes of rat liver; (4) the chloroplast membrane of spinach; (5) the outer segments of bovine photoreceptors; (6) a bovine liver microsomal membrane; and (7) the plasma membrane of bovine erythrocytes. Several hitherto unexplained phenomena, such as the reconstitution of the electron transfer chain and the requirement of lipid for certain enzymic activities, are rationalized in terms of this capability of the repeating units to form membranes. On the basis of the properties of lipid-containing and lipiddepleted membranes, a theory of how membranes are formed has been developed. This theory depends primarily on the restrictions, imposed by phospholipids, on interactions between repeating units. Lipid-containing repeating units can associate into a continuum, one unit or macromolecule thick that can be identified with a membrane. By contrast, lipid-depleted repeating units tend to form a bulk phase, which can be identified with a random three-dimensional polymeric aggregate. Enzymic activities cannot be fully demonstrated when the repeating units exist in the state of a bulk phase.

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. The plasma membrane has been isolated from chick embryo fibroblasts by a procedure involving differential and equilibrium density gradient centrifugations of untreated and zinc-treated cells. 2. 2. The plasma membrane was isolated as large membrane sheets in the case of the zinc-treated cells and as smaller membrane vesicles and fragments from the untreated cells. Phosphohydrolases including the (Na + +K + )-stimulated Mg 2+ -ATPase (EC 3.6.1.3) and CTPase (EC 3.6.1.4) were concentrated in the plasma membrane fraction. This fraction was essentially free of RNA and the intracellular and mitochondrial membrane enzymes, NADH-cytochrome c reductase (EC 1.6.2.1) and succinate dehydrogenase (EC 1.3.99.1). 3. 3. Sialic acids, cholesterol and phospholipid were concentrated in the plasma membrane preparations. The latter two components accounted for 65% by weight of the membrane.

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.