Proteins in tissue extracts which bind insulin-like growth factor binding protein-3 (IGFBP-3).
Membrane associated IGFBP-3 is now known to play a role in the modulation of IGF at the cellular level, but mechanisms involved in cell membrane binding are far from certain. In this study we report the identification and initial structural characterisation of proteins in a range of sheep and rat tissues which specifically bind recombinant human non-glycosylated IGFBP-3. Tissues were homogenised in Tris HCl (0.1 M, pH 7.4), containing proteolytic enzyme inhibitors and the residues re-extracted in buffer containing SDS and Triton X-100 (both 1% w/v) prior to analysis. These were subjected to SDS-PAGE, electro-blotted onto nitrocellulose and subjected to ligand blot analysis (LBA) using radioiodinated IGFBP-3 as ligand. LBA revealed a major band of binding activity migrating at 60 kDa in extracts of rat muscle while sheep muscle contained forms of 52 and 40 kDa as the principal species and ovine pancreatic extracts an abundance of the 40 kDa variant alone. Distribution of the binding activity appears tissue specific. Apart from skeletal muscle, pancreas and a small amount of the 52 kDa form in pituitary, analysis revealed no evidence of IGFBP-3 binding in a range of other sheep tissues including liver, kidney, spleen, intestine, adrenal, brain, mammary, uterus, ovary and plasma. The binding activity is TCA precipitable, trypsin digestible, dose responsive and relatively specific for IGFBP-3 since the related proteins recombinant human IGFBP-2 and purified caprine IGFBP-4 failed to bind.(ABSTRACT TRUNCATED AT 250 WORDS)
Hodgkinson
S
S
AgResearch, Ruakura Agricultural Research Centre, Hamilton, N.Z.
Fowke
P
P
al Somai
N
N
McQuoid
M
M
D000818:Animals; D001345:Autoradiography; D002352:Carrier Proteins / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D018971:Insulin-Like Growth Factor Binding Protein 2; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D018482:Muscle, Skeletal / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D013002:Somatomedins / Q000378:metabolism*
D002352:Carrier Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D013002:Somatomedins; D006493:Heparin