Purification and characterization of human adrenomedullin binding protein-1.
We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45 degrees C for 5 d in solution or at 100 degrees C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-alpha and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.
Qiang
Xiaoling
X
The Feinstein Institute for Medical Research, Manhasset, New York, USA.
Wu
Rongqian
R
Ji
Youxin
Y
Zhou
Mian
M
Wang
Ping
P
D000818:Animals; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D006470:Hemorrhage / Q000150:complications / Q000473:pathology / Q000517:prevention & control; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D007511:Ischemia / Q000473:pathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008107:Liver Diseases / Q000209:etiology / Q000473:pathology; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D044967:Serum / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018932:Chemokine CCL2; D017242:Complement Factor H; D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; C491766:Ccl2 protein, rat; C493495:CFH protein, human