title: Surface-induced alterations in the kinetic pathway for cleavage of human high molecular weight kininogen by plasma kallikrein.
abstract: We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO4) and sulfatides. Using a combination of SDS-polyacrylamide gel electrophoresis, Western blotting with polyclonal antibodies that specifically recognize the COOH terminus of the bradykinin moiety, and high pressure liquid chromatography analyses of the cleavage reaction, we have identified two intermediates in the formation of bradykinin from intact kininogen and demonstrated that alternative cleavage pathways are followed in the absence and presence of surfaces. The COOH-terminal bradykinin cleavage occurred first both in the absence and presence of DxSO4, producing a 103-kDa HK intermediate consisting of disulfide-linked heavy and light chains that retained the kinin moiety. In the presence of DxSO4, this was followed exclusively by the NH2-terminal bradykinin cleavage and release of kinin with no apparent change in molecular mass. Subsequently, a slower cleavage of an 8-kDa peptide from the amino terminus of the HK light chain occurred to form a 95-kDa end product. In contrast to this sequential cleavage pattern, NH2-terminal bradykinin and light chain cleavages occurred randomly in the absence of DxSO4, resulting in the production of an additional 95-kDa intermediate that retained bradykinin but had lost the 8-kDa peptide from the HK light chain. Comparison of the relative rates of the three kallikrein cleavages in the absence and presence of DxSO4 indicated that the surface enhanced the rates of both bradykinin cleavages 2-4-fold, but inhibited the light chain cleavage rate approximately 10-fold, thereby accounting for the change from a partially random to a sequential cleavage pattern in the presence of the surface. Steady-state kinetic analysis revealed that DxSO4 enhanced the kcat/KM for bradykinin release by the rate-limiting NH2-terminal bradykinin cleavage by approximately 2-fold due exclusively to an increase in kcat. Sulfatides appeared to produce the same effects on the pattern of HK cleavages as DxSO4. Blocking of the nonactive site, i.e. exosite, interaction between kallikrein and HK with excess prekallikrein or a synthetic peptide containing the region of HK that interacts with the kallikrein exosite significantly reduced the rate of bradykinin release as well as HK cleavages detected by SDS-polyacrylamide gel electrophoresis either in the absence or presence of DxSO4, indicating that the exosite interaction facilitates bradykinin cleavage.
authors.affiliation: Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202.
authors.forename: M A
authors.initials: MA
authors.lastname: Tayeh
authors.forename: S T
authors.initials: ST
authors.lastname: Olson
authors.forename: J D
authors.initials: JD
authors.lastname: Shore
mesh_terms: D001798:Blood Proteins / Q000378:metabolism; D001920:Bradykinin / Q000096:biosynthesis* / Q000276:immunology; D002413:Cations, Divalent / Q000494:pharmacology; D016264:Dextran Sulfate / Q000494:pharmacology; D006801:Humans; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D007704:Kininogens / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008954:Models, Biological; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D013433:Sulfoglycosphingolipids / Q000494:pharmacology; D013499:Surface Properties
chemical_list: D001798:Blood Proteins; D002413:Cations, Divalent; D007704:Kininogens; D008670:Metals; D010446:Peptide Fragments; D013433:Sulfoglycosphingolipids; D016264:Dextran Sulfate; D007610:Kallikreins; D001920:Bradykinin