Hepatocellular cytokeratins as substrates of transglutaminases.
To examine whether hepatocellular cytokeratins can serve as substrates of transglutaminases (TG) TG-catalyzed incorporation of [3H]putrescine into, as well as cross-linking of, cytokeratins was studied. Purified guinea pig liver TG and mouse liver TG present in 105,000 x g supernatants were used as enzymes. Isolated mouse liver cytokeratin filaments, heterotypic tetramers (A2D2), as well as cytokeratin filaments reconstituted from isolated and column-purified liver cytokeratin polypeptides A and D served as substrates. Moreover, to more closely mimic the situation within the cell, mouse liver homogenate containing cytokeratins was also used. Cross-linked proteins were identified as cytokeratins by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytokeratin antibodies. The results indicated that mouse liver cytokeratins can serve as substrates of homologous and heterologous TG. However, liver cytokeratin components A and D differed in this respect. Depending on the experimental conditions either components D or components A were better substrates of TG-mediated cross-linking as revealed by increased high molecular weight aggregates, which failed to enter the gels, concomitant with a decrease of the monomer band suggesting a more intimate relationship between homologous cytokeratin polypeptides within the filament. The results presented provide the basis for studies of TG-induced cross-linking of cytoskeletal components in hepatocytes that may occur during liver cell injury associated with increased intracellular Ca2+ concentrations.
Zatloukal
K
K
Division of Molecular Pathology, University of Graz School of Medicine, Austria.
Denk
H
H
Lackinger
E
E
Rainer
I
I
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D015151:Immunoblotting; D007382:Intermediate Filaments / Q000187:drug effects / Q000378:metabolism; D007633:Keratins / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D051379:Mice; D008815:Mice, Inbred Strains; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D011503:Transglutaminases / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology
D010455:Peptides; D007633:Keratins; D011503:Transglutaminases