Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand‐Derivative Stain with a FITC‐Kisspeptin Derivative

ChemMedChem ◽  
2020 ◽  
Vol 15 (18) ◽  
pp. 1699-1705
Author(s):  
Koki Hasegawa ◽  
Rika Maedomari ◽  
Younosuke Sato ◽  
Kumiko Gotoh ◽  
Shinji Kudoh ◽  
...  
Keyword(s):  
Ligand Blot ◽  
Western Ligand Blot

scholarly journals Corrigendum: Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand‐Derivative Stain with a FITC‐Kisspeptin Derivative

ChemMedChem ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. 725-725
Author(s):  
Koki Hasegawa ◽  
Rika Maedomari ◽  
Younosuke Sato ◽  
Kumiko Gotoh ◽  
Shinji Kudoh ◽  
...  
Keyword(s):  
Ligand Blot ◽  
Western Ligand Blot

Most of the circulating insulin-like growth factors-I and -II are present in the 150 kDa complex during human pregnancy

1991 ◽  
Vol 131 (3) ◽  
pp. 491-497 ◽  
Author(s):  
S. E. Gargosky ◽  
P. C. Owens ◽  
P. E. Walton ◽  
J. A. Owens ◽  
J. S. Robinson ◽  
...  
Keyword(s):  
Growth Factors ◽  
Pregnant Women ◽  
Blot Analysis ◽  
Binding Proteins ◽  
Size Exclusion ◽  
Igf I ◽  
Ligand Blot ◽  
Igfbp 3 ◽  
Insulin Like Growth Factors ◽  
Western Ligand Blot

ABSTRACT The aim of this study was to assess the molecular size distribution of insulin-like growth factors (IGFs) complexed to IGF-binding proteins (IGFBPs) in serum of non-pregnant and pregnant women. Sera were fractionated on a size-exclusion column at pH 7·4 to resolve different IGF–IGFBP complexes from unbound IGFs. Each fraction was further chromatographed on a size-exclusion column under acid conditions to dissociate IGFs from binding proteins prior to measurement of the IGF content of the complexes. The IGF-containing fractions from the acid column were assayed specifically for IGF-I and IGF-II. Serum pooled from pregnant women contained more IGF-I and IGF-II than serum from non-pregnant women. In both groups most of the IGF-I and IGF-II was found in a large (150 kDa) complex in serum and the remainder was present in complexes eluting in the 40–50 kDa region at pH 7·4. Some free IGF-I was also detected. Serum fractions collected by size-exclusion chromatography at pH 7·4 were also analysed by Western-ligand blotting to characterize the IGFBPs in the two main IGFBP size classes. In serum pooled from non-pregnant women, the 150 kDa IGF–IGFBP complexes contained a 40–50 kDa IGFBP doublet following Western-ligand blot analysis. This IGFBP co-migrated with a pure IGFBP-3 standard. IGFBPs of 34, 28 and 24 kDa all contributed to the 40–50 kDa IGF–IGFBP complexes of the pH 7·4 chromatograph. In serum pooled from pregnant women, the 40–50 kDa IGFBP-3 doublet and 34 kDa IGFBP were not evident in any fractions from the pH 7·4 column. Thus, although the amounts of IGF-I and IGF-II in 150 kDa IGF–IGFBP complexes were increased during late pregnancy, IGFBP-3 measured by Western-ligand blot analysis of this complex was greatly diminished. The large amount of IGF-I and IGF-II in 150 kDa complexes is strong evidence for the presence of IGFBP-3 in serum during pregnancy, because IGFs and IGFBP-3 are normally present in equimolar amounts in this complex. We suggest that during pregnancy in women, the IGFBP-3 in the 150 kDa complex becomes unstable and this may explain the failure of the Western-ligand blot to detect IGFBP-3. Journal of Endocrinology (1991) 131, 491–497

Influence of nutrition on hepatic IGF-I mRNA levels and plasma concentrations of IGF-I and IGF-II in meat-type chickens

1996 ◽  
Vol 149 (1) ◽  
pp. 181-190 ◽  
Author(s):  
K Kita ◽  
F M Tomas ◽  
P C Owens ◽  
S E Knowles ◽  
B E Forbes ◽  
...  
Keyword(s):  
Binding Protein ◽  
Protein Diet ◽  
Low Protein Diet ◽  
Low Protein ◽  
Igf I ◽  
Initial Control ◽  
Ad Libitum ◽  
Ligand Blot ◽  
Meat Type ◽  
Western Ligand Blot

Abstract We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards. In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and β-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to β-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations. Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition. IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment. Journal of Endocrinology (1996) 149, 181–190

Lipoprotein lipase binding to adipocytes: evidence for the presence of a heparin-sensitive binding protein

1993 ◽  
Vol 265 (6) ◽  
pp. E880-E888 ◽  
Author(s):  
A. Sasaki ◽  
P. Sivaram ◽  
I. J. Goldberg
Keyword(s):  
Cell Surface ◽  
Lipoprotein Lipase ◽  
Binding Protein ◽  
Surface Protein ◽  
Surface Binding ◽  
Cell Membrane Proteins ◽  
Adipocyte Cell ◽  
Capillary Endothelial Cells ◽  
Fold Excess ◽  
Ligand Blot

Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.

Ligand Blot Analysis of Insulin-Like Growth Factor-Binding Proteins using Biotinylated Insulin-Like Growth Factor-I

1997 ◽  
Vol 49 (1) ◽  
pp. 1-7 ◽  
Author(s):  
J. Grulich-Henn ◽  
J. Grulich-Henn ◽  
S. Spiess ◽  
S. Spiess ◽  
U. Heinrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Blot Analysis ◽  
Binding Proteins ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Factor I ◽  
Growth Factor I ◽  
Ligand Blot

scholarly journals Structural requirements for the interaction between tissue factor and factor VII: characterization of chymotrypsin-derived tissue factor polypeptides

1993 ◽  
Vol 292 (1) ◽  
pp. 7-12 ◽  
Author(s):  
D P O'Brien ◽  
J S Anderson ◽  
D M A Martin ◽  
P G H Byfield ◽  
E G D Tuddenham
Keyword(s):  
Tissue Factor ◽  
High Performance ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Coagulation Factor Vii ◽  
Chromogenic Assay ◽  
Membrane Bound ◽  
Ligand Blot

Tissue Factor (TF) is the cellular receptor for coagulation Factor VII/VIIa (FVII/VIIa). TF binds to FVIIa and promotes the rapid activation of the zymogen substrates Factors IX and X (FIX and FX) to the respective serine proteinases. In order to probe structure-function relationships in TF, we have subjected the truncated membrane-bound variant, TF 1-243, to proteolytic digestion in SDS-containing gels. Three major polypeptide fragments were generated by proteolysis of TF 1-243 with chymotrypsin, producing cleavages C-terminal to residues 34, 76 and 103. All three polypeptides, TF 35-243, 77-243 and 104-243, bound biotinylated human FVII in a highly specific ligand blot assay. High-performance electrophoretic chromatography was used to isolated chymotrypsin-derived fragments of TF. These purified fragments bound FVII in ligand blots, and two of the three polypeptides exhibited much reduced, but significant, procoagulant activity in a chromogenic assay for the generation of Factor Xa in the presence of FVIIa and Ca2+. The smallest chymotrypsin-derived TF polypeptide, TF 104-243, showed reduced binding of FVII in ligand blot analyses, inhibited the activity of the full-length molecule, but had no procoagulant activity. These data suggest that a part of the binding site for FVII is contained within the TF sequence 104-243. The sequence TF 1-34 either contains a part of the FVII-binding domain or its removal leads to dysfunctional folding, disrupting binding sites elsewhere in the molecule.

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