A DNase I binding/immunoprecipitation assay for actin.
An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.
D000199:Actins / Q000032:analysis*; D000818:Animals; D001792:Blood Platelets / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases / Q000276:immunology; D004720:Endonucleases / Q000276:immunology; D005260:Female; D006801:Humans; D008099:Liver / Q000032:analysis; D009131:Muscle, Smooth, Vascular / Q000032:analysis; D009132:Muscles / Q000032:analysis; D009206:Myocardium / Q000032:analysis; D010053:Ovary / Q000032:analysis; D010063:Ovum / Q000032:analysis; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D012617:Sea Urchins
D000199:Actins; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I