A set of stage-dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines.

A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two-dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.

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.