Translation of messenger RNA from canine tracheal epithelial cells: identification of mucin core protein.
A high-molecular-weight mucin (Mr approximately 11.0 x 10(6)) was purified from canine tracheal pouch secretions. The mucin was deglycosylated by treatment with trifluoromethane sulfonic acid for 8 h at 8 degrees C and subsequently with alpha-N-acetylgalactosaminidase. These treatments almost completely removed the carbohydrate moieties. The amino acid compositions of the deglycosylated and native mucins were similar, indicating that the deglycosylation procedure used did not cause notable degradation of the protein core. Antiserum specific for deglycosylated canine tracheal mucin was produced by immunization of rabbit with the antigen. RNA was isolated from fresh canine tracheal epithelial cells by extraction with guanidine isothiocyanate/hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ RNA. The poly(A)+ RNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine and [3H]leucine as radiolabels. The translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum to deglycosylated mucin. A labeled product of molecular weight 72,000 was present in the immunoprecipitate. When canine liver poly(A)+ RNA was used as control, no radioactivity above background was detected in the immunoprecipitate. It is concluded that the primary translation product of the canine tracheal epithelial cells is a 72,000-D protein and the monomer subunit of the mucin is about 167,000 D. Thus, in the native state, the canine tracheal mucin consists of several associating subunits.
Virmani
A K
AK
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Shankar
V
V
Gilmore
M S
MS
Graves
D C
DC
Sachdev
G P
GP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002474:Cell-Free System; D004285:Dogs; D004848:Epithelium / Q000502:physiology; D008970:Molecular Weight; D009077:Mucins / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014132:Trachea / Q000502:physiology*
D000596:Amino Acids; D009077:Mucins; D012333:RNA, Messenger