Immunochemical studies of human high molecular weight kininogen and of its complexes with plasma prekallikrein or kallikrein.

D.M Kerbiriou,B.N Bouma,J.H Griffin

doi:10.1016/s0021-9258(19)85618-1

Abstract

Human plasm contains at least two distinct kininogens, designated high Mr and low Mr kininogens, that differ in their Mr and susceptibility to different kallikreins. By limited proteolysis, plasma kallikrein cleaves high Mr kininogen to liberate kinin and gives a molecule containing two disulfide-linked polypeptide chains. Following reduction and alkylation of the two-chain molecule, the heavy chain and the light chain were isolated, and antisera were raised in goats against the alkylated heavy and light chains. The anti-heavy chain antiserum immunoprecipitated both high Mr and low Mr kininogen, whereas the anti-light chain antiserum was specific for high Mr kininogen. Thus, the heavy chain of kinin-free high Mr kininogen and low Mr kininogen extensively share identical immunologic determinants. In contrast, the immunologic determinants of the isolated light chain are unique to high Mr kininogen. Using immunochemical techniques, it was shown that high Mr kininogen or the light chain derived from it forms a complex either with prekallikrein or with kallikrein. Titrations of prekallikrein or kallikrein with increasing amounts of either high Mr kininogen or its alkylated light chain indicated that the complexes contain equimolar amounts of each molecule. These results show that a single site for prekallikrein or kallikrein binding to high Mr kininogen resides in the light chain region of the molecule.

Highlights

Differintheir M, and susceptibility to different kal- Under some conditions, high M, kininogen co-purifies or colikreinsB. ylimited proteolysis, plasmakallikrein precipitates with Factor XI [23] or prekallikrein [24, 25]
Thheeavy negatively charged surfaces [13] promotes the binding of lZ5Ilabeled Factor XI or prekallikrein to negatively charged surfaces [26]. These observations suggest that high M, kininogen plays a role as a surface receptor for Factor XI and prekallikrein at the sites of their activation by surface-bound activated Factor XI1 (Hageman factor)
Using immunoshow that a single site for prekallikrein or kallikrein electrophoretic techniques, we demonstrate that high M, kinbinding to high Mr kininogen resides in the light chain inogen or its isolated alkylated light chain is able to form region of the molecule

Summary

MATERIALS AND METHODS

Hered to this column and was eluted as described elsewhere [32] This simplified procedure yields prekallikrein preparations in samples were hydrolyzed in vacuo with 6 N HCI at 105°Cfor 24 h and which small amounts of high M , protein, presumably y-globulins, analyzed using a Beckman 121M amino acid analyzer [37].The were occasionallyobserved on nonreduced SDS gels. These proteins protein concentration of the samples was determined from the total were removed by an immunoadsorption technique as follows. The serum was applied at 4°C to a column (1.5 X 5 cm) containing Sepharose to which the

RESULTS

Low MW Kininogen

LightChain Preincubated With

DISCUSSION

Heavy Chain Inactive