Unstable inter-H chain disulfide bonding and non-covalently associated J chain in rat dimeric IgA.
Disulfide bonds are a major force in stabilizing the three-dimensional structure of immunoglobulins. To determine the pattern of interchain disulfide bonding between the four H chains, four L chains and single J chain of rat dimeric IgA (dIgA), we analyzed dIgA from the LO DNP-64 hybridoma by diagonal SDS-PAGE. Bands corresponding to one, two, three and four H chains, one and two L chains and the free J chain were observed under non-reducing conditions, suggesting that the interchain disulfide bonds in rat dIgA are unstable under denaturing conditions. Similar patterns of disulfide bonding were observed in three other hybridoma or myeloma dIgAs from LOU/CN rats. In contrast, when dIgA pretreated with iodoacetamide (IA) was analyzed by the same technique, only bands corresponding to four H chains, one and two L chains and the free J chain were observed, suggesting that blocking free sulfhydryl groups stabilizes the inter-H chain disulfide bonds. Reaction of dimeric LO DNP-64 dIgA with 5,5'-dithiobis-(2-nitrobenzoic acid) or with 14C-IA demonstrated that this dIgA contains an average of 4 moles of free sulfhydryl groups per mole of protein under non-denaturing conditions and 9 moles of free sulfhydryl groups under denaturing conditions. Taken together, the results suggest that interchain disulfide bonds in rat dIgA are unstable, presumably due to the influence of nearby free sulfhydryl groups, and that non-covalent forces are critical for stabilizing the dIgA complex. The results also indicate that J chain is entirely non-covalently associated with the H chains, an apparently unique feature of rat dIgA. A model for interchain disulfide bonding in rat dIgA is proposed.
Chintalacharuvu
K R
KR
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106.
Lamm
M E
ME
Kaetzel
C S
CS
D000818:Animals; D004220:Disulfides; D015180:Electrophoresis, Gel, Two-Dimensional; D007070:Immunoglobulin A / Q000737:chemistry* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007133:Immunoglobulin Joining Region / Q000737:chemistry*; D008956:Models, Chemical; D051381:Rats
D004220:Disulfides; D007070:Immunoglobulin A; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region