Influence of carrier-specific, thymus-derived cells on the immunologlobulin M antibody response to staphylococcal lipoteichoic acid

The immunoglobulin M antibody response to the lipoteichoic acid (LTA) of Staphylococcus aureus ATCC 6538P was examined by a procedure in which erythrocytes sensitized with periodate-activated LTA were used for the detection of immunoglobulin M-producing plaque-forming cells LTA-specific plaque-forming cells were first detected 2 days after immunization with heat-killed bacterial cells, and maximal numbers of plaque-forming cells, mostly of the immunoglobulin M class rather than the immunoblogulin G or immunoglobulin A class, were attained by day 4; specificity for LTA was affirmed by plaque inhibition tests. No plaque-forming cells were found in mice given isolated LTA over a 10,000-fold range of immunizing doses. Mice pretreated with a carrier known to activate thymus-derived helper lymphocytes produced a plaque-forming cell response to LTA only when immunized with LTA bound to the same carrier. This suggests that carrier-specific thymus-derived cells are needed to initiate an antibody response to poorly immunogenic LTA. Since an antibody response can be elicited in mice given heat-killed cells, other cell wall and/or cell membrane constituents may play an important role as immunologically active carriers for this antigen.     

Purification and some properties of maleylpyruvate hydrolase and fumarylpyruvate hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn(2+) ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.