Protease Resistance and Conformation of Laminin

Laminin, obtained from a tumor basement membrane, was treated with neutral proteases (trypsin, chymotrypsin, elastase, subtilisin, Stuphylococcus aureus protease), which all produced similar fragment patterns upon prolonged digestion. The patterns were different from those obtained for fibronectin, which showed a much higher susceptibility to proteolysis by the same enzymes. Four large fragments could be purified which accounted together for more than half of the mass of laminin. They were found to differ in size, amino acid composition, spectral properties and antigenicity. The largest fragment 1 (M r 260000‐300000) was rich in half‐cystine (120 residues/1000) and showed a circular dichroism spectrum indicating the absence of α helix and β structure. Fragment 3 (M r = 50000) possessed beta; structure and was able to bind onto heparin‐Sepharose. Fragments 2 (M r = 50000) and 4 (M r = 75000) were related structures and their relative yields depended on the protease used. They showed spectra similar to those of fragment 1. Electron microscopy revealed that fragment 1 consists of three rodlike elements (length 26 nm) connected to each other at one end. The other fragments appeared as globules (fragment 3), short rods (fragment 2) or globules connected to a short rod (fragment 4). Data obtained from limited proteolysis indicated that fragment 1 and 4 (or 2) are in close proximity to each other in the three short arms of laminin, which in its intact form has the shape of an asymmetric cross. The long arm appeared to be readily degraded by proteases. Circular dichroism studies of native laminin indicated about 55%, aperiodic structures, 15%β structure and 30%× helix. The α helix was readily destroyed by proteolysis and showed a sharp, reversible transition at 58° C. Stability of these structures was decreased by reduction of disulfide bonds or by increasing concentrations of guanidine. Heat denaturation rendered laminin susceptible to plasmin, which did not degrade the native protein. Cleavage occurred mainly within the 440000‐M r polypeptide chain of laminin and was accompanied by a partial loss of the long arm.