EXTRACTION OF CYTOKERATIN FROM THE HUMAN SUBMANDIBULAR-GLAND AND ITS ELECTROPHORETIC ANALYSIS

We evaluated a method for extracting cytokeratin (CK) from the normal human submandibular gland and analyzed CK distribution by one- and two -dimensional electrophoresis. Four submandibular glands without histol ogical changes under a light microscope were used as specimens. Interm ediate filamentous protein was extracted by a prior modified method CK was not adequately extracted in 1-2% sodium dodecyl sulfate - 5% 2-me rcaptoethanol solution. On the other hand, 11 bands with molecular wei ghts of 40-58 K were obtained after extraction in 9.5 M urea - 5% 2-me rcaptoethanol solution. Although fragmentation of cytoskeletal protein was observed, there were no differences in bands according to the str ength of homogenization. This fragmentation seemed to be due to restri ction degradation by protease. Immunoblotting revealed the reaction of this CK extract to 2 pan-CK antibodies, i.e., K8.13, which recognizes type II basic CK, and C-ll, which recognizes CK4, CK5, CK6, CK8, CK10 , CK13, CK18. Among monospecific antibodies, anti-CK7 (LDS-68), CK8 (M 20), CK13 (KS-1A3), CK18 (CY-90), and CK19 (BA17) antibodies showed po sitive reactions. Glial fibrillary acidic protein, neurofilament (NF) 68 K, NF160 K, or NF200 K showed no reactions. These results of one- a nd two-dimensional electrophoretic analysis suggest the presence of CK 5, CK7, CK8, CK13, CK14, CK15, CK17, CK18, and CK19 in the normal subm andibular gland.