Association of actin with DNA and nuclear matrix from Guerin ascites tumour cells.

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro 125I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices - 5 micrograms/ml in nuclei, of which 50% are bound to DNA and 30% being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCl) to DNA and represents a component of the internal nuclear matrix.

Possible synthesis of steroid-protein for immunization with a fixed narrow range of the hapten-protein ratio.

A comparison of two methods for synthesis of a steroid-protein conjugate (progesterone-11 alpha-hemisuccinate-BSA) has been made. Under the conditions of the method described, using tetrahydrofurane as a medium for the coupling reaction, stable intermediate products were obtained. The resulting conjugate had a narrow range of the hapten-protein ratio (18-20 steroid molecules per molecule of BSA), very good solubility in water and almost no unreacted BSA in the sample. Antisera raised against this conjugate, applied in low dose (50 micrograms), had quite satisfactory characteristics concerning their titre and specificity. The tests for recovery, sensitivity and the range of measurement established the possibility of using such antisera for the radioimmunoassay of progesterone.

The effect of the purity of the iodinated tracer on the specificity of a homologous assay of ovine follicle stimulating hormone.

Six different chromatographic procedures were employed to separate the intact labelled ovine follicle stimulating hormone after iodination of a highly purified preparation. The immunoreactivity of the fractions was tested in the conditions of a double-antibody radioimmunoassay. A single point crossreaction was introduced to calculate the interference of luteinizing hormone in the assay. The results obtained after SDS electrophoresis of the hormone were compared with the results of the autoradiography of the fractions subjected to electrophoresis after labelling and purification. Intact hormone with both subunits present was recovered following chromatography on Blue Sepharose C1-6B or high resolution gel filtration on Ultrogel AcA-54.