Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk.
By means of gel filtration on a TSK-gel HPLC column in the presence of 8 M urea, a 37-kDa polypeptide (p37) was completely separated from lactoferrin (LF) in the heparin HII fraction of the partially purified LF fraction prepared from bovine milk. Purified p37 was identified as a fibroblast growth factor-binding protein (FGF-BP), since its N-terminal 14 amino acid residues (KKEGRNRRGSKASA) were 100% identical to the corresponding sequence of bovine FGF-BP. It was found, in vitro, that (i) p37 had a higher binding affinity with bFGF than bLF; (ii) p37 functioned as a phosphate acceptor for at least three protein kinases (PKA, CK1 and CK2); (iii) bLF stimulated about 3-fold the PKA-mediated phosphorylation of p37, but suppressed its phosphorylation by CK1; and (iv) galloyl pedunculagin was an effective inhibitor for the phosphorylation of p37 by PKA and CK1. Furthermore, the physiological correlation between p37 and bLF may be regulated through specific phosphorylation of p37 by PKA, since p37 fully phosphorylated by PKA did not bind to bLF in vitro. The sulfatide-induced conformational changes in p37 enabled the phosphorylation of p37 by CK1 and also reduced its ability to bind with bLF in vitro. From these results presented here, it is concluded that (i) p37 (FGF-BP) may be tightly associated with bLF in bovine milk; and (ii) the physiological correlation between p37 and bLF may be regulated by the PKA-mediated full phosphorylation of p37 or by the direct binding of sulfatide to p37 in vivo.
Kawakami
Akio
A
Laboratory of Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, Kitasato 1-15-1, Sagamihara 228-8555, Japan.
Hirayama
Kyoko
K
Kawakami
Fumitaka
F
Kawakami
Hiroshi
H
Fujihara
Michio
M
Ohtsuki
Kenzo
K
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D047389:Casein Kinase I / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007781:Lactoferrin / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D013433:Sulfoglycosphingolipids / Q000494:pharmacology
D002352:Carrier Proteins; D008894:Milk Proteins; D013433:Sulfoglycosphingolipids; D047389:Casein Kinase I; D047390:Casein Kinase II; D017868:Cyclic AMP-Dependent Protein Kinases; D007781:Lactoferrin