POTENTIAL MOLECULAR CHAPERONES INVOLVED IN LAMININ CHAIN ASSEMBLY

To explore potential molecular chaperones involved in the intracellula r assembly of laminin chains, bovine aortic endothelial cells were tre ated with a thiol cleavable divalent cross-linking reagent, dithio-bis -(succinimidylpropionate), and cellular proteins cross-linked to lamin in chains were co-immunoprecipitated with anti-laminin antiserum. Sodi um dodecylsulfate (SDS) gel electrophoresis of the precipitate under r educing condition showed polypeptides with estimated sizes of 80, 60 a nd 50 kDa together with laminin chains. Two dimensional electrophoresi s, in which non-reducing and reducing SDS electrophoresis were combine d, suggested that many molecules of these polypeptides were cross-link ed to each laminin chain. Sepharose CL-4B beads conjugated with E8 fra gment of mouse laminin-l was prepared. Affinity chromatography with th e beads of microsomal proteins from rat liver showed that Bip and HSP7 0 associated to laminin chains and dissociated upon ATP hydrolysis. Pr otein-disulfide isomerase also showed affinity to the column. GRP94 an d calnexin showed strong affinity and were washed out only with a dete rgent solution. Thus, many molecular chaperones are suggested to be in volved in the intracellular assembly of laminin chains.