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J F Perdue

Publications and source records attributed to J F Perdue.

At least 19 recordsLinked to original sources
PubMed1992

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.

Amino Acid Sequence↗
PubMed1992

Glucose utilization in a patient with hepatoma and hypoglycemia. Assessment by a positron emission tomography.

Tumor glucose use in patients with non-islet-cell tumors has been difficult to measure, particularly in hepatoma, because of hepatic involvement by neoplasm. We studied a patient with nonhepatic recurrence of hepatoma after successful liver transplantation. Tumor tissue contained messenger RNA for insulin-like growth factor-II (IGF-II), and circulating high molecular weight components and E-peptide of IGF-II were increased. Glucose use measured by isotope dilution with [3-3H]glucose was 7.94 mg/kg fat-free mass per min, and splanchnic glucose production was 0.93 mg/kg fat-free mass per min. Glucose uptake and glucose model parameters were independently measured in tissues by positron emission tomography with 18F-fluoro-2-deoxy-D-glucose. Glucose uptake by heart muscle, liver, skeletal muscle, and neoplasm accounted for 0.8, 14, 44, and 15% of total glucose use, respectively. Model parameters in liver and neoplasm were not significantly different, and glucose transport and phosphorylation were twofold and fourfold greater than in muscle. This suggests that circulating IGF-II-like proteins are partial insulin agonists, and that hypoglycemia in hepatoma with IGF-II production is predominantly due to glucose uptake by skeletal muscle and suppression of glucose production.

Adult↗
PubMed1991

The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.

3T3 Cells↗
PubMed1991

Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors.

In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (appMr) of 15,000 precursor form of human insulin-like growth factor-II (hIGF-II) was shown to be greater than that of completely processed hIGF-II for human fetal-derived fibroblasts, and both were more potent than rIGF-I. Since it is generally acknowledged that the stimulation of cell replication by the IGFs is mediated by IGF-I receptors, we undertook to determine whether differences between the receptors' affinity for the two Mr forms of hIGF-II and recombinant IGF-I (rIGF-I) or between its efficiency to couple specific growth factor occupancy to the activation of protein kinase could explain the greater replicating potential of appMr 15,000 hIGF-II. Equilibrium dissociation, i.e. Kd, and inhibition, i.e. Ki, constants were determined by measuring the ability of rIGF-I, hIGF-II, appMr 15,000 hIGF-II, insulin, and the antireceptor monoclonal antibody alpha IR-3 to compete with 125I-labeled rIGF-I and hIGF-II for binding to purified preparations of IGF-I receptors prepared from an enriched source of fetal membrane, i.e. human term placenta. The results of these experiments established that 1) hIGF-II and appMr 15,000 hIGF-II bind to the IGF-I receptor with the same affinity as rIGF-I, e.g. with Kd and Ki values between 0.03-0.07 nM; 2) the total binding capacity, i.e. Ro, for IGF-I binding was not statistically different from the Ro calculated for IGF-II binding; and 3) the statistical analysis of 12 data sets from the competitive binding experiments for goodness of fit indicated that a 1-site model for IGF-I and -II binding was a better fit of the data than a 2-site model. Measurements of the stimulation of IGF-I receptor autophosphorylation at low ligand concentrations established that appMr 15,000 hIGF-II and hIGF-II were more effective than rIGF-I in coupling receptor occupancy to the activation of its protein kinase. At saturating ligand concentrations, the 3 had similar potencies. The original preparation of appMr 15,000 hIGF-II contains a mixture of forms with acidic isoelectric points (pIs) and was more potent than Mr 7,500 IGF-II in stimulating receptor autophosphorylation. These results are consistent with the relative potencies of this preparation, hIGF-II, and rIGF-I in stimulating the replication of 12-week-old fetal dermal fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1989

Purification and characterization of an insulin-like growth factor II variant from human plasma.

An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.

Amino Acid Sequence↗
PubMed1989

Expression of insulin-like growth factor-II mRNA in fetal kidney and Wilms' tumor. An in situ hybridization study.

The pattern of insulin-like growth factor II (IGF-II) mRNA expression in developing kidney and Wilms' tumor was examined with in situ hybridization. In developing kidney, IGF-II was primarily expressed in blastemal cells and lost with their differentiation. In triphasic Wilms' tumor, a similar relationship was found. But in a monomorphous Wilms', tumor cells with epithelial differentiation expressed IGF-II mRNA. These data suggest that IGF-II may be involved in fetal nephrogenesis, that its expression is inversely coupled to normal epithelial differentiation, and that this differentiation may be aberrantly regulated in Wilms' tumor.

Child, Preschool↗
PubMed1988

Insulin-like growth factor II mRNA expression in human breast cancer.

Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observations that show IGF-II is expressed in mesenchyme. These findings suggest that in breast cancer IGF-II is produced by stromal tissue elements and potentially by the malignant epithelial cells. Therefore, IGF-II may function as an autocrine or a paracrine growth factor in different breast tumors.

Blotting, Northern↗
PubMed1987

Presence of somatomedin receptors on primary human breast and colon carcinomas.

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.

Breast Neoplasms↗
PubMed1987

Purification and characterization of a unique high molecular weight form of insulin-like growth factor II.

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.

Amino Acid Sequence↗
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↗
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↗