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...
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| Veröffentlicht in: | The Journal of biological chemistry 1981-06, Vol.256 (12), p.6291-6295 |
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| container_title | The Journal of biological chemistry |
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| creator | M C Snabes A E Boyd, 3rd R L Pardue J Bryan |
| description | 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. |
| doi_str_mv | 10.1016/S0021-9258(19)69161-1 |
| format | Article |
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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.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(19)69161-1</identifier><identifier>PMID: 6263912</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Actins - analysis ; Animals ; Blood Platelets - analysis ; Cricetinae ; Cricetulus ; Deoxyribonuclease I ; Deoxyribonucleases - immunology ; Endonucleases - immunology ; Female ; Humans ; Liver - analysis ; Muscle, Smooth, Vascular - analysis ; Muscles - analysis ; Myocardium - analysis ; Ovary - analysis ; Ovum - analysis ; Precipitin Tests ; Rabbits ; Rats ; Sea Urchins</subject><ispartof>The Journal of biological chemistry, 1981-06, Vol.256 (12), p.6291-6295</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c410t-12e550ab9cf8ab6dd71e847e721ab61016f9bf6b5a4593161908e2d1030855093</citedby><cites>FETCH-LOGICAL-c410t-12e550ab9cf8ab6dd71e847e721ab61016f9bf6b5a4593161908e2d1030855093</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,778,782,27907,27908</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/6263912$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>M C Snabes</creatorcontrib><creatorcontrib>A E Boyd, 3rd</creatorcontrib><creatorcontrib>R L Pardue</creatorcontrib><creatorcontrib>J Bryan</creatorcontrib><title>A DNase I binding/immunoprecipitation assay for actin</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>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.</description><subject>Actins - analysis</subject><subject>Animals</subject><subject>Blood Platelets - analysis</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Deoxyribonuclease I</subject><subject>Deoxyribonucleases - immunology</subject><subject>Endonucleases - immunology</subject><subject>Female</subject><subject>Humans</subject><subject>Liver - analysis</subject><subject>Muscle, Smooth, Vascular - analysis</subject><subject>Muscles - analysis</subject><subject>Myocardium - analysis</subject><subject>Ovary - analysis</subject><subject>Ovum - analysis</subject><subject>Precipitin Tests</subject><subject>Rabbits</subject><subject>Rats</subject><subject>Sea Urchins</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1981</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEtLAzEQx4MotVY_QmFBED2szSSbbHIs9VUoelDBW8juZttI92Gyi_Tbmz7o1bkMw_z-8_gjNAZ8Dxj45B1jArEkTNyCvOMSOMRwgoaABY0pg69TNDwi5-jC-28cIpEwQANOOJVAhohNo4dX7U00jzJbF7ZeTmxV9XXTOpPb1na6s00dae_1JiobF-m8s_UlOiv12purQx6hz6fHj9lLvHh7ns-mizhPAHcxEMMY1pnMS6EzXhQpGJGkJiUQyu0XpcxKnjGdMEnDAxILQwrAFIsglHSEbvZzW9f89MZ3qrI-N-u1rk3Te5Uyzjlh_F8QGJVJkogAsj2Yu8Z7Z0rVOltpt1GA1fYitfNVbU1TINXOVwVBNz4s6LPKFEfVwcjQv973V3a5-rXOqMw2-cpUKpyngARQAv0DfKZ8kg</recordid><startdate>19810625</startdate><enddate>19810625</enddate><creator>M C Snabes</creator><creator>A E Boyd, 3rd</creator><creator>R L Pardue</creator><creator>J Bryan</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>19810625</creationdate><title>A DNase I binding/immunoprecipitation assay for actin</title><author>M C Snabes ; A E Boyd, 3rd ; R L Pardue ; J Bryan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c410t-12e550ab9cf8ab6dd71e847e721ab61016f9bf6b5a4593161908e2d1030855093</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1981</creationdate><topic>Actins - analysis</topic><topic>Animals</topic><topic>Blood Platelets - analysis</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Deoxyribonuclease I</topic><topic>Deoxyribonucleases - immunology</topic><topic>Endonucleases - immunology</topic><topic>Female</topic><topic>Humans</topic><topic>Liver - analysis</topic><topic>Muscle, Smooth, Vascular - analysis</topic><topic>Muscles - analysis</topic><topic>Myocardium - analysis</topic><topic>Ovary - analysis</topic><topic>Ovum - analysis</topic><topic>Precipitin Tests</topic><topic>Rabbits</topic><topic>Rats</topic><topic>Sea Urchins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>M C Snabes</creatorcontrib><creatorcontrib>A E Boyd, 3rd</creatorcontrib><creatorcontrib>R L Pardue</creatorcontrib><creatorcontrib>J Bryan</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>M C Snabes</au><au>A E Boyd, 3rd</au><au>R L Pardue</au><au>J Bryan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A DNase I binding/immunoprecipitation assay for actin</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1981-06-25</date><risdate>1981</risdate><volume>256</volume><issue>12</issue><spage>6291</spage><epage>6295</epage><pages>6291-6295</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>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.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>6263912</pmid><doi>10.1016/S0021-9258(19)69161-1</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0021-9258 |
| ispartof | The Journal of biological chemistry, 1981-06, Vol.256 (12), p.6291-6295 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_75666256 |
| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Actins - analysis Animals Blood Platelets - analysis Cricetinae Cricetulus Deoxyribonuclease I Deoxyribonucleases - immunology Endonucleases - immunology Female Humans Liver - analysis Muscle, Smooth, Vascular - analysis Muscles - analysis Myocardium - analysis Ovary - analysis Ovum - analysis Precipitin Tests Rabbits Rats Sea Urchins |
| title | A DNase I binding/immunoprecipitation assay for actin |
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