Biochemical characterization of proteins that co-purify with class II antigens of the murine MHC.
Careful analysis of affinity-purified class II molecules (Ia Ag) from the murine MHC revealed the existence of a set of associated molecules that consistently co-purified with the Ia Ag. SDS-PAGE revealed that molecules of Mr of 41 to 43 kDa and 56 to 58 kDa were associated with the affinity-purified I-Ak Ag from the AKR B cell lymphoma AKTB-1b. Two-dimensional electrophoresis (IEF vs SDS-PAGE) allowed further characterization of four molecules in the 41- to 43-kDa range and two in the 56- to 58-kDa range. All co-purifying proteins had isoelectric points between 5.2 and 6.2. The specificity of the association of the co-purifying molecules with the I-Ak Ag was established by using two criteria. First, with the exception of actin, proteins co-purifying with the I-Ak molecule were not found in samples of affinity-purified class I (H-2Kk) Ag or membrane Ig from the AKTB-1b lymphoma. Second, the use of the amino group-reactive homobifunctional cross-linker 3,3'-dithiobisproprionimidate with crude membranes from AKTB-1b increased the relative amount of materials co-purifying with I-Ak. The use of the membrane-impermeant cross-linker 3,3'-dithiobis(sulfosuccinimidyl) proprionate provided evidence that the interaction between I-Ak and one or more of the co-purifying components occurs on the cytoplasmic face of the membrane. Two of the co-purifying molecules have been identified. The major material in the 41- to 43-kDa range was partially sequenced, leading to its identification as cytoplasmic actin. One of the components in the 56- to 58-kDa range was tentatively identified as one of the isozymes (RII) of the regulatory subunit of the cAMP-dependent protein kinase, based on the use of the photoaffinity label 8-azido-cAMP.
Newell
M K
MK
Division of Basic Immunology, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
Justement
L B
LB
Miles
C R
CR
Freed
J H
JH
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification; D008223:Lymphoma / Q000032:analysis; D008285:Major Histocompatibility Complex; D008565:Membrane Proteins / Q000145:classification / Q000302:isolation & purification*; D051379:Mice; D008806:Mice, Inbred AKR; D009363:Neoplasm Proteins / Q000302:isolation & purification; D011494:Protein Kinases / Q000302:isolation & purification
D000199:Actins; D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D000949:Histocompatibility Antigens Class II; D007527:Isoenzymes; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011494:Protein Kinases