Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.
Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.
Brown
J C
JC
Advanced Drug Delivery Research Unit, Ciba-Geigy Pharmaceuticals, Horsham, West Sussex, U.K.
Spragg
J H
JH
Wheeler
G N
GN
Taylor
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005696:Galactosidases; D006031:Glycosylation; D006801:Humans; D007106:Immune Sera; D007797:Laminin / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid; D015017:Yolk Sac / Q000032:analysis*; D001616:beta-Galactosidase
D007106:Immune Sera; D007797:Laminin; D011993:Recombinant Fusion Proteins; D005696:Galactosidases; D001616:beta-Galactosidase