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

1984 ◽  
Vol 62 (11) ◽  
pp. 1237-1245 ◽  
Author(s):  
James F. Perdue
Keyword(s):  
Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Receptors ◽  
Cross Reactivity ◽  
Blood Levels ◽  
Protein Kinase Activity ◽  
Binding Studies ◽  
Normal Children ◽  
Igf I

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.

Purification of a basic somatomedin, from human plasma Cohn fraction IV-1, with physicochemical and radioimmunoassay similarity to somatomedin-C and insulin-like growth factor

1979 ◽  
Vol 57 (11) ◽  
pp. 1289-1298 ◽  
Author(s):  
R. Marvin Bala ◽  
B. Bhaumick
Keyword(s):  
Growth Factor ◽  
Human Plasma ◽  
Protein Separation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Molecular Size ◽  
Protein Band ◽  
Insulin Like Growth Factor ◽  
Igf I

A basic somatomedin (SM) was purified from human plasma Cohn fraction IV-1 using an initial acid–ethanol–acetone extraction procedure followed by alternating molecular size or charge protein separation techniques. The final recovery of SM bioactivity was approximately 2% of that present in the starting Cohn fraction. The purified SM has an approximate molecular weight of 7500, pI 8.6, 4000 SM bioactivity units per milligram (as measured by a hypophysectomized rat bioassay) and a parallel approximately equipotent radioimmunoassay dose–response curve to SM-C and insulin-like growth factor-I (IGF-I). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of this purified SM revealed a single protein band. The preliminary determination of the amino acid sequence of the N terminus suggested that this SM preparation was over 75% pure and the first five N-terminal amino acids were identical with those of IGF-I.

Comparison of receptors for insulin-like growth factor II from various rat tissues

1987 ◽  
Vol 115 (1) ◽  
pp. 35-NP ◽  
Author(s):  
J. E. Taylor ◽  
C. D. Scott ◽  
R. C. Baxter
Keyword(s):  
Growth Factor ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcutaneous Fat ◽  
Cross Reactivity ◽  
Rat Tissues ◽  
Insulin Like Growth Factor ◽  
Major Band ◽  
Factor Ii

ABSTRACT Receptors for insulin-like growth factor II (IGF-II) have been compared in solubilized microsomal membranes from rat lung, brain, kidney, heart, epididymal and subcutaneous fat, ovary, testis and adrenals. Highest binding/μg protein was seen with testicular membranes. Receptors from all tissues showed high affinity for human IGF-II (mean association constant = 65 litres/nmol) and a high degree of specificity (mean IGF-I cross-reactivity 0·3%; no cross-reactivity with insulin). Affinity labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed binding was only to a type-II IGF receptor, with a major band seen at a molecular weight of about 230 000 in lung, brain, kidney and testis, and 240 000 in heart, fat and adrenal gland. All tissues showed broad or bimodal pH dependence of binding, with optima seen at about pH 6 and pH 9. Mild stimulation of IGF-II binding by low calcium concentrations (1–2 mmol/l) was seen in all tissues, although higher concentrations were inhibitory in the brain. It was concluded that IGF-II receptors from different rat tissues, when studied under uniform conditions, show similar binding affinities but differences in size and regulation which might be missed if receptors are examined in separate studies. J. Endocr. (1987) 115, 35–41

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells

1992 ◽  
Vol 262 (4) ◽  
pp. F639-F646 ◽  
Author(s):  
A. V. Cybulsky ◽  
P. R. Goodyer ◽  
M. D. Cyr ◽  
A. J. McTavish
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Egf Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activation ◽  
Scatchard Analysis ◽  
Glomerular Epithelial Cells ◽  
Epidermal Growth

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

scholarly journals Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA

2019 ◽  
Vol 25 (3) ◽  
pp. 310-319
Author(s):  
Yuan Dong ◽  
Hanjin Hou ◽  
An Chen ◽  
Wei Ma ◽  
Moli Yin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Enzyme Hydrolysis ◽  
Clinical Samples ◽  
Sds Page ◽  
Thrombotic Diseases ◽  
D Dimer

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

scholarly journals Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Characterization of Proteins in the Outer Membrane Preparation of a Murine Pathogen, Helicobacter bilis

2001 ◽  
Vol 69 (5) ◽  
pp. 3502-3506 ◽  
Author(s):  
Zhongming Ge ◽  
Peter Doig ◽  
James G. Fox
Keyword(s):  
Outer Membrane ◽  
Wide Spectrum ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Membrane Preparation ◽  
Laboratory Animals ◽  
Helicobacter Bilis ◽  
H Pylori

ABSTRACT Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins fromH. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

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