Laminin-nidogen complex

Extraction with chelating agents and structural characterization

Authors


Correspondence to R. Timpl, Abteilung Bindegewebsforschung, Max-Planck-Institut für Biochemie, D-8033 Martinsried bei München, Federal Republic of Germany

Abstract

Large quantities of intact laminin-nidogen complex could be extracted from a mouse tumor basement membrane with a physiological buffer containing EDTA. Analysis of the purified complex demonstrated that the two proteins occur in an equimolar ratio and that anchoring of these complexes to the extracellular matrix requires divalent cations. Reversible dissociation of the complex was achieved with 2 M guanidine · HCl and had been used for purification of the individual components. Electron microscopy and binding studies using laminin fragments demonstrated that nidogen interacts specifically with the center of the cross-shaped laminin moleculle as represented by the short-arm structure fragment 1. The complex was also useful to confirm and refine a previously proposed dumb-bell structure of nidogen and to prepare and characterize the cell-binding fragment 8 from the long arm of laminin.

Abbreviations
PhMeSO2F

phenylmethylsulfonyl fluoride

MalNEt

N-ethylmaleimide

EHS tumor

Engelbreth-Holm-Swarm tumor

Ancillary