It is proposed to analyze the composition and structural organization of basement membranes. Structural macromolecules derived from basement membranes (type IV collagen, type V collagen, laminin, fibronectin) will be isolated from human placenta, calf lung and the murine EHS-sarcoma by salt extraction and limited enzyme treatment. The extracted proteins will be purified by differential salt precipitation, ion exchange and gel permeation chromatography and characterized by polyacrylamide electrophoresis, peptide fingerprinting techniques, the rotary shadowing technique and immunochemically. Monoclonal antibodies to the isolated proteins or fragments will be produced by somatic cell hybridization technique and these antibodies will be used as tools to determine structural heterogeneity of basement membrane (type IV, V) collagens of isolated material by immunopreciptation and of a variety of basement membranes in vivo by immunohistochemical techniques. These antibodies will be also used to study the expression of components present in basement membranes in tissue culture systems in order to understand the possible contribution of various cells to basement membrane synthesis and turnover in normal and possible alterations in pathological conditions. Preliminary evidence suggests, that type IV collagen is organized by forming a 3-dimensional network, in which one end each of four native molecules is combined to form a cross-linked structure, while the other end of each molecule participates in a different cross-linked structure. This organization is drastically different from the arrangement of interstitial collagens into fibillar arrays. We would like to study the molecular details of the basic framework and its possible interaction with other macromolecules which have been suggested by us and other groups to exist within basement membranes: type V collagen and laminin.
