PROTEINS IN TISSUE-EXTRACTS WHICH BIND INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 (IGFBP-3)

被引:20
作者
HODGKINSON, S
FOWKE, P
ALSOMAI, N
MCQUOID, M
机构
[1] AgResearch, Ruakura Agricultural Research Centre, Hamilton
关键词
D O I
10.1677/joe.0.145R001
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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) prier 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. Additionally, IGFBP-3 binding to these species appeared unaffected by presaturation of IGFBP-3 with IGF-L nor by their transient acidification which together with molecular weight estimates and their pattern of tissue distribution suggests they are not related to the acid labile subunit of the ternary 150 kDa IGFBP complex. IGEBP-3 binding and molecular mobility of the species were unaffected by sample reduction but binding was suppressed in a concentration dependent fashion by heparin. Finally LBA using I-125-Concanavalin A revealed the 40 kDa species to be glycosylated while the 52 kDa form was not. Functions of these novel proteins in targeting or modulating IGF activity at the cellular level remain to be clarified.
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页码:R1 / R6
页数:6
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