Laminin, a major structural glycoprotein of basement membranes, has been found to self-associate in vitro into large polymers. The formation of these complexes can be followed by the development of turbidity upon incubation in neutral phosphate buffer at 21-35° C and is seen to be time-, concentration-, and temperature-dependent. The process is thermally reversible at 4°C and the protein can be cycled between a dispersed and an aggregrated state by alternating between 4 and 35°C. Following incubation at 35°C much of the monomeric laminin, which sediments at 11.4 S, is now seen to sediment at greater than 25 S. Both by turbidometric and sedimentation analysis, an apparent critical concentration for assembly of about 0.1 mg/ml (10-7 M) is observed and is interpreted as evidence for a nucleation-prolongation polymerization mechanism. The relative paucity of intermediates seen in a size-distribution analysis lends further support for this model. On platinum replicas obtained by rotary shadowing analysis, mostly free monomers are seen in the cold while after incubation at 35°C, large multimeric aggregates with smaller amounts of oligomers are observed. The interaction between individual molecules appears to be specific because the dimers, trimers, and smaller oligomers are only associated at the terminal glomular domains of the laminin molecules. In addition, removal of the globular domains of laminin with pepsis, which yields fragment P1, abolishes self-association. A divalent cation dependency for polymerization can be demonstrated and incubation in the presence of EDTA stops the polymerization at an oligomeric intermediate step. Hence overall laminin self-assembly can be divided into at least two steps: an initial temperature-dependent, divalent cation independent step followed by a divalent cation-dependent step.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|