Isolation of a subunit of laminin and its role in molecular structure and tumor cell attachment.
Laminin, the glycoprotein of basement membranes, migrates as two components of 200 kilodaltons (kDa) (alpha subunit) and 400 kDa (beta subunit) after reduction on polyacrylamide gel electrophoresis. We have isolated the alpha subunit and studied its structure by electron microscopy and its function as an attachment factor for tumor cells. Using selective proteolysis of laminin by alpha-thrombin, the beta subunit was removed without any change in the quantity or size of the alpha subunit. Removal of the beta subunit caused a 35-40% decrease in the total mass of the laminin molecule. The alpha and beta subunits differed by 50-fold in the amount of reducing agent required for complete migration on polyacrylamide gels. By electron microscopy, the whole laminin molecule appeared as a "cross" with three identical short arms (37 nm) and one long arm (75 nm). The alpha subunit examined by electron microscopy was missing the long arm and had no change in the length of the three short arms. This subunit of laminin mediated the attachment of human squamous carcinoma cells to type IV collagen. Such attachment properties were lost after pepsin treatment which is known to remove the globular end regions of the short arms. We conclude that the beta subunit of laminin is embodied in the long arm of the molecule and that the alpha subunit consists of three similar chains of 200 kDa. The globular end regions of the laminin short arms are required for the attachment of tumor cells to type IV collagen.
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D002460:Cell Line; D055598:Chemical Phenomena; D002621:Chemistry; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007797:Laminin; D051379:Mice; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000302:isolation & purification; D009374:Neoplasms, Experimental / Q000032:analysis; D013917:Thrombin
D006023:Glycoproteins; D007797:Laminin; D009363:Neoplasm Proteins; D003094:Collagen; D013917:Thrombin