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J F Perdue

Publications and source records attributed to J F Perdue.

At least 37 records · Page 2Linked to original sources
PubMed1984

Insulin-like growth factor II receptors. Molecular radius and molecular weight determination using quantitative polyacrylamide gel electrophoresis.

High resolution quantitative polyacrylamide gel electrophoresis was employed under nondenaturing conditions to calculate a molecular weight, Mr, for rat placental membrane receptors that bind insulin-like growth factor II (IGF-II). An n-octylglucoside-soluble extract of receptors that had been enriched 20-fold during Sephacryl S-300 gel chromatography and designated peak 1 (Perdue, J. F., Chan, J. K., Thibault, C., Radaj, P., Mills, B., and Daughaday, W. H. (1983) J. Biol. Chem. 258, 7800-7811) was incubated with 125I-IGF-II in the presence or absence of an excess of unlabeled IGF-II and electrophoresed in glass tubes containing highly cross-linked polyacrylamide gels at concentrations ranging from 5 to 12% (w/v). A specifically labeled peak of IGF-II binding activity was identified by freezing, slicing, and counting the gels. The proteins eluted from the region of the gel when cross-linked to 125I-IGF-II with disuccinimidyl suberate and electrophoresed in the presence of sodium dodecyl sulfate have the same molecular weight, Mr, as the previously described IGF-II receptor. From the slope of a plot of log of the relative mobility, RF, at each of 6 to 7 gel concentrations for the 125I-IGF-II-receptor and for each of seven standard proteins (Ferguson-Hedrick plot), a retardation coefficient, KR, was determined. Using a reference curve constructed from a plot of square root KR versus the molecular radius, R, of the standard proteins, the IGF-II receptor was estimated to have a R of 4.13 nm and a calculated Mr of 250,000. Thus, quantitative procedures that separate native proteins based on their size and charge have provided information which is in good agreement with the results of studies of the IGF-II-receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and supports the premise that it is a monomeric single chained glycoprotein, constrained by intradisulfide bonds and with a mass of 250 kDa.

Animals↗
PubMed1984

Chemistry, structure, and function of insulin-like growth factors and their receptors: a review.

Human serum contains two classes of somatomedins (Sm) with similar three-dimensional structures and mass (i.e., approximately 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: monoclonal and polyclonal antibodies to insulin receptors recognize determinants on receptors that bind IGF-I; 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Acromegaly↗
PubMed1983

The biochemical characterization of detergent-solubilized insulin-like growth factor II receptors from rat placenta.

A membrane preparation, the R3, obtained by differential centrifugation of rat placental homogenates is enriched in receptors that bind insulin-like growth factor II (IGF-II) preferentially and with avidity (Daughaday, W.H., Mariz, I.K., and Trivedi, B. (1981) J. Clin. Endocrinol. Metab. 53, 282-288). When this preparation was incubated with 2% (w/v) octyl-beta-D-glucopyranoside for 60 min at 0-4 degrees C, 60% of the membrane protein was solubilized without loss of binding activity. The 125I-IGF-II binding properties of the detergent-solubilized receptors were found to be similar to those of the membrane-associated receptor. The rate constants for association, ka, and dissociation, kd, and equilibrium dissociation constant, KD, were 8.5 X 10(8) M-1 min-1, 7.5 X 10(-3) min-1, and 1.3 nM for the detergent-solubilized receptors and 5.3 X 10(8) M-1 min-1, 4.2 X 10(-3) min-1, and 0.6 nM for the membrane receptors. Gel chromatography on Sephacryl S-300 concentrated the solubilized receptors into a major peak of binding activity with a Stokes radius of 7.2 nm; a second peak of less specific binding had a Stokes radius of 4.3 nm. The receptors in the major peak bound 125I-IGF-II with a KD of 0.6 nM; the total binding capacity, Ro, was 21.6 pmol mg of protein-1 compared to 1.6 pmol mg of protein-1 for the membrane-associated receptor. Centrifugation of the receptors on 5-20% (w/v) gradients of sucrose in H2O or D2O disclosed a heterogeneous pattern of receptor distribution. When they were labeled with 125I-IGF-II prior to centrifugation, a major form of the receptor with a sedimentation constant, S20,w, of 9.9 X 10(13) s and other, possibly smaller, forms of the receptor were observed. However, only the 9.9 s20,w form of the receptor was observed if it was labeled with 125I-IGF-II subsequent to centrifugation. Based on these hydrodynamic measurements and a partial specific volume of 0.72 cm3/g, the IGF-II receptor was calculated to have a Mr of 290,000 and frictional ratio, f/fo, of 1.6. This value for the Mr is similar to the mass of 220,000 or 250,000 Dal determined by cross-linking 125I-IGF-II to the membrane- or detergent-solubilized receptors with disuccimidyl suberate and separating the complex by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels in the absence or presence of dithiothreitol, respectively.

Animals↗
PubMed1981

Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin.

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.

Animals↗
PubMed1980

Stereospecific D-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts.

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.

Animals↗
PubMed1980

An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors.

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 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

Animals↗
PubMed1980

In cultured chick embryo fibroblasts the hexose transport components are not the 75 000 and 95 000 dalton polypeptides synthesized following glucose deprivation.

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.

Animals↗
PubMed1979

Failure to confirm evidence for a nonvirion tumor-specific surface antigen in avian retrovirus-transformed cells.

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.

Animals↗
PubMed1977

Immunologic identification of fetal calf serum-derived proteins on the surfaces of cultured transformed and untransformed rat cells.

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 polyacrylamide 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 98,000 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.

Animals↗
PubMed1977

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts.

At 5 microgram/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1-20 micrometer). hydrogen peroxide (500 micrometer), and methylene blue (50 micrometer) mimic the effect of insulin in CEF. PCMB-S,1 a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1-1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80-100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not -L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.

Amino Acids↗
PubMed1976

Mitogen receptors in chick embryo fibroblasts. Kinetics, specificity, unmasking, and synthesis of 125I-insulin binding sites.

Insulin, a mitogen for cultured chick embryo fibroblasts (Temin, H.M. (1968) Cancer 3, 771-787), has been employed to characterize the effects of mitogen/cell membrane interactions as it relates to growth. The specific binding of 125I-insulin to substratum-attached cells is time- and temperature dependent and is optimum at a pH of 7.0. Fetal calf and chicken sera, somatomedin "A/C mixed," and desalanine or native porcine insulin compete with 125I-insulin for membrane-binding sites. Proinsulin, although competing less effectively than native insulin for binding, is more effective than desoctapeptide insulin. Unrelated polypeptide hormones do not compete for 125I-insulin binding. The lowest concentration of insulin at which specific binding is detected is 0.1 nM. Scatchard plot analysis of the binding data indicates that there are two types of binding sites in confluent cultures of fibroblasts: one of high affinity (K1 = 2 to 6 X 10(8) M-1) and low capacity, the other of low affinity (K2 = 0.8 to 3.0 X 10(7) M-1) and high capacity. Approximately 1.9 and 7.1 X 10(3) molecules of insulin are bound at each site, respectively. A 10-min incubation at 24 degrees of the fibroblasts with 10 mug/ml of trypsin causes a 2-fold stimulation of specific 125I-insulin binding and a similar 2-fold increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation. Neuraminidase treatment also produces a 37% increase in specific 125I-insulin binding but treatment with alpha-chymotrypsin or phospholipase C are without significant effect. The results of this and additional experiments support the hypothesis that trypsin treatment of chick embryo fibroblasts leads to an unmasking of 125I-insulin binding sites. Serum starvation of fibroblasts for 12 or 24 h produces a 2.5- to 5-fold increase in specific 125I-insulin binding. This increase is the result of an increase in the number of hormone-binding sites from 9 X 10(3) to 6 X 10(4) per cell which are predominantly of the low affinity type. There is no change in the affinity constants. The presence of camptothecin, or cordycepin, or cycloheximide in the incubation medium completely blocks the increase in number of 125I-insulin-binding sites resulting from serum starvation. The addition of native insulin to the medium of serum-starved cultures also blocks this increase. The magnitude of insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation correlates with the levels of occupancy of the low affinity 125I-insulin-binding sites in untreated fibroblasts. In fibroblasts cultured in the absence of serum, the marked increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation parallels the increase in number of mitogen receptors. The concentration of insulin that produces a half-maximum stimulation of thymidine incorporation is calculated to be 5 X 10(-8) M. At this concentration of insulin, 42% of the receptor sites are occupied.

Animals↗
PubMed1976

Regulation of sugar transport in chick embryo fibroblasts and in fibroblasts transformed by a temperature-sensitive mutant of the Rous sarcoma virus.

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.

Animals↗
PubMed1976

Loss of the post-translational control of nutrient transport in vitro and in vivo virus-transformed chicken cells.

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.

Aminoisobutyric Acids↗
PubMed1976

The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses.

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 compexes 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 degrees 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.

Animals↗