PURIFICATION OF FIBRILLIN-CONTAINING MICROFIBRILS AND COLLAGEN-VI MICROFIBRILS BY DENSITY GRADIENT CENTRIFUGATION

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M-r microfi bril-rich preparations isolated from nuchal ligament by bacterial coll agenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin wi thin the gradient was achieved by SDS-PAGE/Western blotting. Large col lagen VI microfibrillar aggregates were present at the top of the grad ient. Hyaluronidase pretreatment dissociated these aggregates and enab led purification of collagen VI microfibrils at a density of 1.33 g/ml . Fibrillin-containing microfibrils separated at 1.37 g/ml and copurif ied with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolec ules provides a powerful means of addressing their molecular compositi on, organization, and structure:function relationships. (C) 1998 Acade mic Press.