Purification of heparin-binding epidermal growth factor-like growth factor from pig uterine luminal flushings, and its production by endometrial tissues.
Pig uterine luminal flushings contain at least four heparin-binding growth factors (HBGF) that stimulate fibroblast [3H]thymidine incorporation. One of these factors, which appeared to be a relatively minor HBGF, was eluted from heparin affinity columns by 1.0 M NaCl and was found to compete with 125I-epidermal growth factor (EGF) for binding to an endometrial carcinoma cell line. This EGF receptor (EGF-R)-binding property was abolished by an antiserum to heparin-binding EGF-like growth factor (HB-EGF) that specifically blocks binding of HB-EGF to the EGF-R. Reverse-phase HPLC resulted in the purification of two EGF-R-binding activities correlated with 13,500 and 17,000 M(r) proteins that reacted with an antiserum raised against residues 9-26 of human HB-EGF. Uterine extracts also contained an EGF-R-binding factor that was eluted from heparin by 1.0 M NaCl and was antagonized by HB-EGF antiserum. Endometrial mRNA subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR through the use of HB-EGF-specific primers yielded fragments of the predicted size. Cloning of the nested PCR product revealed a 380-bp porcine HB-EGF cDNA sequence that was 78-85% homologous to primate or rodent HB-EGF. HB-EGF was immunohistochemically localized primarily to the luminal epithelium in both pregnant and nonpregnant animals.
Kim
G Y
GY
Department of Surgery, Ohio State University College of Medicine and Children's Hospital, Columbus 43205, USA.
Besner
G E
GE
Steffen
C L
CL
McCarthy
D W
DW
Downing
M T
MT
Luquette
M H
MH
Abad
M S
MS
Brigstock
D R
DR
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004717:Endometrium / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000302:isolation & purification; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006493:Heparin / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D011869:Radioligand Assay; D013552:Swine; D013936:Thymidine / Q000378:metabolism; D014599:Uterus / Q000737:chemistry / Q000378:metabolism*
C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D006493:Heparin; D004247:DNA; D066246:ErbB Receptors; D012194:RNA-Directed DNA Polymerase; D013936:Thymidine