Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin
Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo(1(-14)C)acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides.
- Research Organization:
- Children's Hospital National Medical Center, Washington, DC (USA)
- OSTI ID:
- 5879562
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 264:14
- Country of Publication:
- United States
- Language:
- English
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GLYCOPROTEINS
AMINO ACID SEQUENCE
FRACTIONATION
ACETAMIDE
AMINO ACIDS
CARBON 14 COMPOUNDS
ELECTROPHORESIS
LUNGS
MAN
METHYLATION
MOLECULAR WEIGHT
MUCOUS MEMBRANES
PAPAIN
PEPTIDE HYDROLASES
REDUCING AGENTS
THIN-LAYER CHROMATOGRAPHY
TRYPSIN
ULTRACENTRIFUGATION
AMIDES
ANIMALS
BODY
CARBOXYLIC ACIDS
CENTRIFUGATION
CHEMICAL REACTIONS
CHROMATOGRAPHY
ENZYMES
HYDROLASES
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PRIMATES
PROTEINS
RESPIRATORY SYSTEM
SEPARATION PROCESSES
SERINE PROTEINASES
SH-PROTEINASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques