Plasma hyaluronan binding protein (PHBP) is a novel serine protease found in human plasma.1) It cleaved fibrinogen and fibronectin, and converted inactive single chain urokinase type plasminogen activator (scu PA) to the active twochain form (tcu PA).2) PHBP is present in human plasma as a pro-form of 70 kDa and the purified PHBP caused auto-fragmentation to the active hetero-dimer form (50-kDa heavy chain and 27-kDa light chain, both of which are bridged by a disulfide linkage).3) This auto-activation was accelerated in the presence of dextran sulfate and phosphatidylethanolamine.4) The active hetero-dimer further advanced conversion to the inactive form, i.e., the heavy chain to two 26-kDa fragments and the light chain to the 17 and 8-kDa fragments.3) In human plasma, C1 inhibitor is a major inhibitor of PHBP.4) Other groups also reported the serine protease activity of PHBP. [5][6][7][8] In particular, Romisch et al. reported that PHBP activated factor VII of the tissue factor pathway of the coagulation system independently from tissue factor. 5) These results suggest that PHBP is a principal protease in retaining the homeostasis of the body. To study the biological significance of PHBP, we administered to mice CCl 4 which caused hepatic failure, HgCl 2 which injured kidney, and turpentine which led to inflammation, and the induction and activation of PHBP were investigated in this paper. MATERIALS AND METHODS Treatment of MiceCCl 4 (0, 10, 20 or 40% in olive oil, 5 ml/g body weight [b.w.], orally), D-galactosamine (280 mg/ml in phosphate-buffered saline [PBS], 5 ml/g b.w., intraperitoneally), HgCl 2 (1 mg/ml in PBS, 6 ml/g b.w., subcutaneously) or turpentine (5 ml/g b.w., subcutaneously) was administered to an ICR mouse (male, 6-weeks old, Saitama Experimental Animals). Partial hepatectomy (2/3 hepatectomy) was performed according to the methods of Sakamoto et al. 9) and Higgins and Anderson. 10)Preparation of Anti-recombinant-Mouse PHBP Antibody Mouse PHBP expression vector was constructed in pTrcHisC (Invitrogen) and the expression vector was transfected to Escherichia coli (DH5 a, Toyobo).11) The recombinant (r-) mouse PHBP which had His-tag at its N-terminus, was purified from the transformed E. coli with Ni-NTAagarose (QIAGEN) after the cultivation. A rabbit was immunized with 100 mg of purified r-mouse PHBP as an emulsion with Freund's complete adjuvant. Two and three weeks after the first immunization, the rabbit was boosted as above. One week after the final injection, the rabbit was bled and the anti-r-mouse PHBP antiserum was collected after the clotting.Western-Blot Analysis After each treatment of the mouse, the blood was collected from the heart and the plasma was obtained by immediate centrifugation at 1000ϫg for 5 min. Each sample (1 ml) was analyzed on sodium dodecylsulfate-polyacrylaminde gel electrophoresis (SDS-PAGE, 10% polyacrylamide gel). 12) After the electrophoresis, proteins in the gel were electrically transferred to a nitrocellulose filter (Schleicher and Schuell) in 20 mM Tris-...
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