Relationship between tektins and intermediate filament proteins: an immunological study.
Affinity-purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament-like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti-(Lp)-tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti-(Lp)-tektin B cross reacted with two isoforms or different proteins of approximately 54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of approximately 55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC-PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin-specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with alpha-chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti-(Lp)-tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti-(Lp)-tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.
Steffen
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Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
Linck
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D002918:Chymotrypsin; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000276:immunology*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008868:Microtubule Proteins / Q000276:immunology*; D012617:Sea Urchins; D013552:Swine; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D008868:Microtubule Proteins; C044103:tektins; D002918:Chymotrypsin