Immunological Study on the Structural Difference between Ribulose- 1,5-bisphosphate Carboxylase/Oxygenase from Nicotiana tabacum var. John William's Broadleaf and the Tobacco Mutant Su/su

Abstract

Abstract In the present paper we have attempted to characterize by means of immunological methods the molecular difference between ribulose 1,5-bisphosphate carboxylase/oxygenase from the wild type TV. tabacum var. John William's Broadleaf and the tobacco mutant Su/su. The tobacco mutant Su/su exhibits in comparison to the wild type a higher photorespiratory activity. The reagents used in the present study are monospecific antisera to the two bifunctional enzymes to be compared. We have analyzed the oxygenase activity of the two enzymes in dependance on the binding of the amount of antibodies out of the homologous and the not homologous antiserum. These analyses have shown, that the enzymes of both phenotypes were 4 0 % stronger inhibited in the equivalence regions with respect to their oxygenase function by antibodies of the antiserum to ribulose 1,5-bisphosphate carboxylase/oxygenase of the mutant than by antibodies of the wild type antiserum. It should be noted that the antiserum to the mutant enzyme exhibits a 25 % lower antibody titer, than the antiserum to the wild type enzyme. In the region of extreme antibody excess, i.e. when antibodies are mostly monovalently bound and most antigenic determinants are saturated with antibodies, the oxygenase activity of both enzymes decreases towards zero in the presence of the homologous as well as in the presence of non-homologous antibodies. In the region of excess of antigenic determinants, that is when only a few antibody molecules can bind onto the enzymes, only the oxygenase activity of the mutant enzyme is inhibited by its homologous antibodies by 40 % . This apparent difference in the molecular structure of the two bifunctional enzymes to be compared is neither caught in the double immuno diffussion test nor in the tandem crossed immuno electrophoresis, using the two antisera as test reagents. In all cases only fused precipitation bands are observed. Chemical modification of ribulose 1,5-bisphosphate carboxylase/oxygenase by hydroxylamine treatment or treatment with o-(p-nitrophenyl)hydroxylamine or simple heating of the enzyme to 50 °C are immunologically characterized. As a consequence of such treatment considerably less antibodies are adsorbed. The strongest influence exerts a treatment with o-(p-nitrophenyl)-hydroxylamine with simultaneous heat treatment of the enzyme for 20 min at 50 °C.