Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue.
The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.
Clausen
H
H
Biomembrane Institute, University of Washington, Seattle 98119.
White
T
T
Takio
K
K
Titani
K
K
Stroud
M
M
Holmes
E
E
Karkov
J
J
Thim
L
L
Hakomori
S
S
D000017:ABO Blood-Group System / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005646:Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase / Q000032:analysis / Q000097:blood / Q000302:isolation & purification*; D005700:Galactosyltransferases / Q000302:isolation & purification*; D006801:Humans; D008168:Lung / Q000201:enzymology*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D013552:Swine
D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D005700:Galactosyltransferases; D005646:Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase