Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.
A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.
Yae
Y
Y
Clinical Laboratory, Kyushu University Hospital, Fukuoka, Japan.
Inaba
S
S
Sato
H
H
Okochi
K
K
Tokunaga
F
F
Iwanaga
S
S
D000328:Adult; D000596:Amino Acids / Q000032:analysis; D001323:Autoantibodies; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002241:Carbohydrates / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006358:Hot Temperature; D006801:Humans; D007106:Immune Sera / Q000032:analysis*; D037102:Lectins; D008180:Lupus Erythematosus, Systemic / Q000097:blood / Q000276:immunology*; D046911:Macromolecular Substances; D008875:Middle Aged; D008970:Molecular Weight; D011233:Precipitin Tests
D000596:Amino Acids; D001323:Autoantibodies; D001324:Autoantigens; D002241:Carbohydrates; C109662:FCN3 protein, human; D006023:Glycoproteins; D007106:Immune Sera; D037102:Lectins; D046911:Macromolecular Substances