Ligand Blot Procedures Research Articles

Growth rate and changes of the somatotropic axis in beef cattle administered exogenous bovine somatotropin beginning at two hundred, two hundred fifty, and three hundred days of age1

To determine the effects of bovine somatotropin (bST) treatment beginning at 3 ages on the growth rate and components of the somatotropic axis, 40 beef cattle (200 +/- 21 d of age) were randomly assigned to 1 of 4 treatments (10 animals/treatment). Three of the treatment groups received bST (33 mug/kg of BW) daily beginning at 200, 250, or 300 d of age until all animals reached 400 d of age; the fourth group served as controls (0 bST). Animals were housed in pens (5 animals per pen; 2 pens per treatment) and fed a diet formulated for an ADG of 1.2 kg/d. Feed intake (per pen) was measured daily, and BW was determined weekly. Blood samples (10 mL) and ultrasound measurements were collected at 200, 250, 300, 350, and 400 d of age. Serum concentrations of ST and IGF-I were determined by RIA and IGFBP-2 and -3 by ligand blot procedures. Overall, cattle gained 284.0 +/- 14.7 kg of BW with a treatment x week interaction (P < 0.01), such that during the treatment period ADG was 11.6, 8.7, and 15.8% greater (P < 0.05) in cattle treated with bST beginning at 200, 250, and 300 d, respectively, relative to controls during the same time frame. Average DMI was 13.6% less (P < 0.05) in bST-treated cattle than in controls. Increases in ADG coupled with a reduction in DMI resulted in 11.7, 14.0, and 26.4% increases (P < 0.01) in the efficiency of gain (G:F) in bST-treated cattle beginning at 200, 250, and 300 d of age, respectively, compared with contemporary controls. Backfat thickness increased (P < 0.05) over time, but the magnitude of the increase was less in the bST-treated cattle (treatment x week interaction; P < 0.05). Area of the LM increased (P < 0.05) over time but was similar across treatment groups. Serum concentrations of ST, IGF-I, and IGFBP-3 increased (P < 0.05), whereas IGFBP-2 decreased (P < 0.05) over time. The changes in the components of somatotropic axis were more pronounced in bST-treated cattle compared with controls, with the greatest magnitude of response in animals that began bST treatment at 300 d of age. In conclusion, the exogenous bST-induced growth response was greater in animals that began to receive bST administration at 300 d of age and received it for a shorter period (100 d) compared with animals that received bST beginning at 200 or 250 d of age.

Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.

Mapping of a Defined Neurocan Binding Site to Distinct Domains of Tenascin-C

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

Demonstration of membrane estrogen binding proteins in rat brain by ligand blotting using a 17β-estradiol-[ 125I]bovine serum albumin conjugate

This paper describes a ligand blotting procedure to visualize membrane estrogen receptors/binding proteins immobilized on nitrocellulose membranes. Using 17β-estradiol covalently linked with [ 125I]bovine serum albumin (BSA) at the C-6 position (17β-E-6-[ 125I]BSA) as a ligand, three major binding proteins with molecular masses of approximately 23, 28, and 32 kDa were identified from crude synaptosomal fractions (P2) of female rat brains. The binding of 17β-E-6-[ 125I]BSA to these proteins is selective for 17β-estradiol because BSA had no effect, and 17α-E-6-BSA was at least two orders of magnitude less potent than 17β-E-6-BSA in displacing the binding. In addition, [ 125I]BSA and 17α-E-6-[ 125I]BSA at similar concentrations did not bind to these proteins. Competition and saturation assays indicate that the binding affinity of 17β-E-6-[ 125I]BSA for these proteins was in the range of 1–10 nM. These proteins are not contaminants from cytosolic or serum estrogen binding proteins since no corresponding protein bands were found in cytosolic fractions. Three additional protein bands with molecular masses of approximately 18, 40, and 130 kDa were also detected, although inconsistently. The 23 and 40 kDa proteins seem to be concentrated in mitochondrial fractions (mP2), whereas the 28 and 32 kDa proteins are enriched in microsomal fractions (P3). Application of digitonin-solubilized P2 fractions to 17β-estradiol-coupled affinity columns resulted in significant purification of the 23 kDa protein as shown by ligand blotting. This protein was later identified as oligomycin-sensitivity conferring protein (OSCP), as reported previously. These data indicate that specific estrogen binding proteins different from classical nuclear estrogen receptor (66 kDa) are present in the cellular membranes of the female rat brain. The ligand blotting technique described here would also be applicable for the identification of other membrane steroid binding proteins/receptors using similar radiolabelled steroid-BSA conjugates.

A Double Labeling Procedure for Lipoproteins: Independent Visualization of Dual Ligand-Receptor Interaction with Colloidal Gold- and 125I-Labeled Ligands

The analysis of multiple ligand binding to a single receptor molecule poses a methodological challenge. The chicken oocyte 95-kDa receptor for the uptake of the two major yolk lipoprotein precursors very low-density lipoprotein (VLDL) and vitellogenin (VTG) is such a multipotent transport receptor. Here we describe methods for rapid independent and simultaneous analysis of VLDL and VTG binding to this receptor, termed VLDL/VTG receptor. First, further development of a one-step labeling protocol for chicken lipoproteins with colloidal gold (Au) to visualize independently the binding of VLDL and VTG to the chicken VLDL/VTG receptor is reported. The advantage of this protocol is that the preparation of the Au-lipoprotein conjugates is rapid, and utilization of Au-lipoprotein complexes in ligand blots does not require their further purification, while signal enhancement by silver staining is still applicable. Second, the simultaneous use of 125I- and Au-labeled ligands in a one-step ligand blotting procedure facilitates the direct demonstration of the competitive interaction of VLDL and VTG with the receptor. Following sequential processing of the nitrocellulose strips, binding of differently labeled ligands can be studied independently. In summary, we describe a procedure for differential labeling of lipoproteins and its application toward the analysis of receptors that bind more than one ligand.

Visualization of the Chicken Oocyte Lipoprotein Receptor by Ligand Blotting with Biotinylated Plasma and Yolk Very Low Density Lipoproteins

The laying hen 95-kDa oocyte membrane receptor that transports hepatically synthesized very low density lipoprotein (VLDL) and vitellogenin (VTG) from the plasma to growing follicles was visualized by ligand blotting with biotinylated VLDL followed by enhanced chemiluminescence (ECL) detection. Plasma and egg yolk VLDL were isolated by ultracentrifugation and free ∈-amino groups of lysines of apolipoprotein B (apo B), the protein constituent of VLDL that mediates binding to the 95-kDa oocyte membrane receptor, were biotinylated using D-biotin-N-hydroxysuccinimide ester. An apo B concentration of approximately 223 pM was sufficient to give a signal on 2.5 μg of protein from a chicken oocyte membrane detergent extract. Western blotting (immunoblotting) of the laying hen 95-kDa receptor with polyclonal rabbit anti-chicken oocyte VLDL receptor IgG resulted in an ECL signal with the same position of migration as that observed in ligand blots using biotinylated plasma and yolk VLDL. Binding of biotinylated plasma or yolk VLDL to the 95-kDa receptor was abolished by excess unlabeled plasma or yolk VLDL, respectively, as well as by EDTA. Receptor binding activity of biotinylated plasma and yolk VLDL was also demonstrated by a reverse ligand blotting procedure. Compared with conventional techniques involving the use of 125 I-labeled ligands or antibodies, the laying hen 95-kDa oocyte membrane lipoprotein receptor can be safely and rapidly visualized with excellent sensitivity using the present nonradioactive method. In addition, it is suggested that ECL detection can be employed to further study the ligand-binding properties and specificity of this protein, which is essential to vitellogenesis in the chicken.

Presence of Intestinal Lectin-binding Glycoproteins on the Cell Surface ofLactobacillus acidophilus

The presence of intestinal lectin-binding glycoproteins on the cell surface of Lactobacillus acidophilus group lactic acid bacteria was examined. The cell-surface components were extracted with 2 m guanidine hydrochloride, and electrophoresed by SDS–PAGE followed by electroblotting. Several glycoproteins were detected on the blots of the extracts from 7 of the 11 strains tested. The distribution of glycoproteins seemed to be specific for strains rather than for species. β-Galactoside-specific lectin was extracted from chicken intestine, and then was labeled with horseradish peroxidase. The binding of the lectin to glycoproteins detected was assayed by a ligand blotting procedure using the labeled lectin. The lectin-binding glycoproteins estimated to be 60.2 and 43.0 kDa, respectively, were detected from strain L. acidophilus JCM1132T, but not from other strains. The binding was inhibited in the presence of lactose, indicating that the binding was specific. Presumably, the interaction between bacterial cel...

Relationships among concentrations of steroids, inhibin, insulin-like growth factor-1 (IGF-1), and IGF-binding proteins during follicular development in weaned sows.

The experimental objective was to determine how insulin-like growth factor binding proteins (IGFBP), as examined by Western ligand blot procedures, related to porcine follicular steroidogenesis. Weaned sows were ovariectomized at various times after litter removal in three experiments. In experiments 1 and 2, sows were ovariectomized at 48-120 h after weaning. In experiment 1, pools of all small (1-3 mm), medium (greater than 3-6 mm), or large (greater than 6-9 mm) follicles were made for each sow; in experiment 2, fluid was collected individually from the 10 largest follicles per ovary. A third experiment was conducted to examine changes after an ovulatory dose of hCG, but prior to ovulation. In this experiment, sows were treated with eCG at weaning, given hCG 72 h later, and ovariectomized 0-36 h after the ovulatory dose of hCG. Follicular fluid was collected from the 10 largest follicles per sow. In experiments 1 and 2, IGFBP-3 in follicular fluid remained constant over follicle diameters and stage sof development, and IGFBP-2 decreased with advancing follicular development as concentrations of estradiol, androstenedione, and progesterone increased. In experiment 1, after the presumed LH surge when the concentration of all steroids was low, there was a sharp increase in band intensity for IGFBP-2. Similarly, estradiol and androstenedione were low in preovulatory sows in experiment 2, though progesterone increased and IGFBP-2 decreased with follicle diameter. In experiment 3, progesterone remained elevated from 0 to 36 h after hCG, even though IGFBP-2 did not increase until after 24 h post-hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an Insulin‐Like Growth Factor Autocrine‐Paracrine System in the Retinal Photoreceptor‐Pigment Epithelial Cell Complex

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.

Specific binding of plasminogen to vitronectin: Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated K D = 0.4 μM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain.

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.

Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I.

Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.

Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein.

Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and "ligand blotted" with 125I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed.

Partial purification of a high density lipoprotein-binding protein from rat liver and kidney membranes

The existence of a cell receptor which recognises plasma high density lipoprotein (HDL) has been suggested from studies which demonstrate specific binding of HDL 3 to cultured cells derived from various tissues in the body. This study provides evidence of a specific HDL-binding protein in crude plasma membranes prepared from rat kidney and liver. Following separation of solubilised membrane proteins on polyacrylamide gel slabs and ‘Western’ blotting, one major band was identified which bound HDL 3, or apo AI or apo AII The protein, which was present in both liver and kidney membranes, was partially purified by repetitive preparative SDS-polyacrylamide gel electrophoresis and although accompanied by considerable loss of binding activity, could still be detected by the ligand-blotting procedure used initially to detect its presence in cell membranes.

Visualization of heparin-binding proteins by ligand blotting with 125I-heparin

A ligand-blotting procedure which allows detection of heparin-binding proteins is described. Crude commercial heparin was fractionated by chromatography on a column of human plasma low-density lipoproteins immobilized to Sepharose CL-4B. Chromatography yielded an unbound and a bound fraction of heparin, designated URH and HRH, respectively. The HRH fraction was reacted with the N-hydroxysuccinimidyl ester of 3-( p-hydroxyphenyl)propionic acid and then labeled with 125I. Proteins were separated by 3–20% pore-gradient gel electrophoresis, transferred to nitrocellulose, and then assayed for their ability to bind 125I-labeled HRH. Human plasma apolipoproteins B-100, B-48, and E of chylomicrons, very low-density lipoproteins, and low-density lipoproteins bound the 125I-labeled HRH; the radiolabeled heparin did not bind to serum albumin, ferritin, catalase, and lactate dehydrogenase. The ligand-blotting procedure should facilitate the purification of heparin-binding domains from these proteins and, moreover, may be applicable to the investigation of heparin-protein interactions in general.