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A DNase I binding/immunoprecipitation assay for actin.

1981; Elsevier BV; Volume: 256; Issue: 12 Linguagem: Inglês

10.1016/s0021-9258(19)69161-1

1083-351X

Michael C. Snabes, A E Boyd, R L Pardue, Joseph Bryan,

Molecular Biology Techniques and Applications

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/ mol of actin, This I2'I-actin retained the ability to bind to DNase I and inhibit enzymatic activity.The Iz5Iactin-DNase complex can be precipitated by the ad& tion of a monospecific rabbit antibody to DNase I.The efficiency of this immunoprecipitation step i s improved by the use of a second sheep anti-rabbit y-globulin.Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound '251-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 '2SI-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 Fand G-actin in a wide range of cells.A detailed understanding of the role of actin in cellular processes requires a means of quantitating changes in the number and state of assembly of actin molecules.Early assay methods took advantage of the relative abundance of cellular actin and used densitometry of stained electrophoretic gels to study changes in actin content (1-3).Blikstad et al. (4) exploited the unique property of soluble or G-actin to bind to DNase I with high affinity (5, 6) and inhibit the enzymatic hydrolysis of DNA to measure actin.The DNase I inhibition assay measures both soluble (G) and filamentous (F) actin.This procedure requires lengthy individual spectrophotometric determinations of DNA hydrolysis but can reliably measure actin in the microgram/& range.Recently, Morgan et al. ( 7 ) have reported attempts to develop an actin radioim-