Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence.
Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7.5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3' non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 2.6 kb major transcript and two minor transcripts of approximately 2.1 and 1.8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver > kidney > lung >> heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.
Carr
J M
JM
Co-operative Research Centre for Tissue Growth and Repair, Adelaide, Australia.
Grant
P A
PA
Francis
G L
GL
Owens
J A
JA
Wallace
J C
JC
Walton
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013002:Somatomedins / Q000378:metabolism*; D013045:Species Specificity
D002352:Carrier Proteins; D018076:DNA, Complementary; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D013002:Somatomedins