Expression of Insulin-like Growth Factor I (IGF-I) and Its Binding Proteins in Rat Tissues / 대한소아내분비학회지

ABSTRACT

covered with liguid nitrozen and pulverized with a pestle. To the powered tissue 5ml of 3.3M formic acid/0.5% Tween 20 was added and centrifuged at 40,000*g for 10 min. An aliquot of supernate was put into C18 sepak minicolumn to eliminates IGF-BPs. Measurement of IGF-I in rat tissues was done by RIA with anti-hIGF-I antibody and hIGF-I(PSIII) standard which was prepared by Drs. L. E. Underwood and J. J. Van Wyk UNC at Chapel Hill, NC, USA and distributed through the National Hormone and Pituitary Distribution Program. Distribution of IGF-I in rat tissue was seen by SDS-PAGE and ligand blotting method. A cDNA library in lambda gt11 of rat liver was used to isolate the cDNA of IGF-I. Phage containing inserts encoding rat IGF-I were identified by hybridization with biotin labeled synthesized oligomer which was the sequence from 1 to 8 aminoacids of known rat IGF-I. The EcoRI inserts were subcloned into PBluescript SK. The nucleotide sequence of both strands was determined by the dideoxy chain termination method. RESULTS: 1)IGF-BPs in tissue extract which could compete with antibody for IGF-I in measureing the IGF-I were eluted at 50Kdalton molecular weight marker using Protein-pak 300SW column. Using C18-sepak minicolumn, IGF-BPs were completely eliminated from tissue extract as much as possible, using Protein-pak 300SW column. 2)The amount of IGF-I in tissues was as folows liver 575+/-41.6ng/g, lung 552.0+/-40.8ng/g. kidney 503+/-30.8ng/g, heart 449.0+/-30.4ng/g, testis 225+/-18.8ng/g, spleen 146+/-26.4ng/g, muscle 92+/-7.6ng/g and brain 49.0+/-5.8ng/g. The amount of IGF-I in blood was 1403+/-60.8ng/ml. 3)Banding patterns of IGF-BPs in rat tissues extract were obtained using ligand blotting. IGF-BP3 bands at 50 Kdalton molecular weight marker were strongly shown in testis, heart, and lung extracts but not in brain and muscle. IGF-BP1 and 2 band at 30Kdalton molecular weight marker was strongly shown in liver, kidney, spleen, testis, heart and lung. IGF-BP4 band at 21 Kdalton molecular weight marker was weakly shown only in spleen and muscle. 4) The nucleotide sequence of cloned cDNA of rat IGF-I is as follows. 5 10 15 5'----- CC CTT TGC GGG GCT GAG CTG GTG GAC GCT CTT CAG TTC GTG TGT 20 25 30 -GGA CCA AGG GGC TTT TAC TTC AAC AAG CCC ACA GGC TAT GGC- 35 40 45 -TCC AGC ATT CGG AGG GCA CCA CAG ACG GGC ATT GTG GAT GAG------3 CONCLUSION: This study suggests that tissue extraction method for IGF-I from tissues and elimination of IGF-BPs using C18 sepak minicolumn is suitable for measuring in large numbers of samples. Expression of IGF-I and IGF-BPs in multiple tissues suggests some phsiologic function at each tissue level. Subcloning of cDNA of exon 3 and 4 of IGF-I was useful for studying regulation of IGF-IA and IB mRNA in rat tissue.