Purification and lectin-binding properties of s-laminin, a synaptic isoform of the laminin B1 chain.
The extracellular matrix (ECM) at the vertebrate neuromuscular junction is a repository of functionally important molecules, some of which can regulate the formation of synapses during regeneration. One candidate molecule is s-laminin, a 185-kDa homologue of the laminin B1 chain. Whereas several members of the laminin family are present throughout the ECM ensheathing muscle fibers, immunoreactivity for s-laminin is found selectively at synaptic sites in adult and embryonic rats, and is detectable at a time when synaptogenesis is taking place during development. We have reported previously that a rat schwannoma cell line, D6P2T, produces and releases large amounts of s-laminin in culture. We have now purified s-laminin from medium conditioned by these cells by using a simple three-step procedure. Serum-free, conditioned medium is separated by ion-exchange chromatography on DEAE-Sephacel, followed by size-exclusion chromatography on 500 HR-Sephacryl. Finally, s-laminin is dissociated from other ECM components by agarose gel electrophoresis under reducing conditions and recovered in solution by extracting slices of agarose gel. The purified preparation displays one silver-stained band that is recognized by three monoclonal antibodies known to bind to different epitopes on s-laminin. Lectin-binding studies demonstrate that s-laminin is a glycoprotein and bears many of the carbohydrate moieties present on the B1 and B2 chains of laminin. Thus, the three 185-220-kDa members of the laminin family are related in both their protein and carbohydrate domains.
Chiu
A Y
AY
Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Ugozolli
M
M
Meiri
K
K
Ko
J
J
D000818:Animals; D015153:Blotting, Western; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007536:Isomerism; D007797:Laminin / Q000302:isolation & purification* / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D009442:Neurilemmoma / Q000737:chemistry / Q000473:pathology; D013569:Synapses / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003470:Culture Media; D007797:Laminin; D037102:Lectins