Generation, characterization and ELISA of monospecific antibodies against the subunits of a Ca2+-dependent protein kinase and a Ca2+-transport ATPase from rabbit skeletal muscle.
Monospecific precipitating sheep antibodies were generated for the first time against the purified, homogeneous alpha-, beta- and gamma-subunits of the Ca2+-dependent protein kinase, phosphorylase kinase, from rabbit muscle. As reference, antibodies against the holoenzyme and the CA2+-transport ATPase of sarcoplasmic reticulum were induced. In all cases antibody titers could be quantitated (standard error 5-10%) by enzyme-linked immunosorbent assay. Differentiation of antibody binding was achieved by quantitative precipitation and complement fixation assays. In general maximal antibody titers were reached 56 days after primary immunization and high titers (approximately 5000) were maintained for several weeks. Anti-alpha, anti-beta and anti-gamma avidly precipitate the denatured subunits employed as immunogens as well as the native enzyme. No cross-reactivity between antibodies against a specific subunit and any of the other heterologous subunits was demonstrable in double immunodiffusion assays providing no evidence for immunologically identical sites on the alpha-, beta- and gamma-subunits. Since anti-alpha, anti-beta and anti-gamma strongly inhibit enzyme activity, it is likely that they do so primarily by sterically interfering with the binding of the large substrate phosphorylase b (Mr 2.0 X 10(5)) to phosphorylase kinase (Mr 1.3 X 10(6)). It cannot be excluded, however, that anti-beta and anti-gamma bind to the active sites on these 2 subunits.
D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000502:physiology; D000917:Antibody Formation; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000276:immunology*; D011232:Chemical Precipitation; D003168:Complement Fixation Tests; D004797:Enzyme-Linked Immunosorbent Assay; D005779:Immunodiffusion; D008297:Male; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000276:immunology; D012756:Sheep
D000906:Antibodies; D010764:Phosphorylase Kinase; D000252:Calcium-Transporting ATPases