Purification and Characterization of Ceramide-Activated Protein Phosphatases
Ceramide has emerged as a potential regulator of diverse cellular functions, and a few direct targets have been identified for its action including protein kinases and phosphatases. In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizin...
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| Veröffentlicht in: | Biochemistry (Easton) 1998-08, Vol.37 (32), p.11232-11238 |
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| container_issue | 32 |
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| container_title | Biochemistry (Easton) |
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| creator | Galadari, Sehamuddin Kishikawa, Katsuya Kamibayashi, Craig Mumby, Marc C Hannun, Yusuf A |
| description | Ceramide has emerged as a potential regulator of diverse cellular functions, and a few direct targets have been identified for its action including protein kinases and phosphatases. In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purified to near homogeneity using hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS−polyacrylamide gel electrophoresis which comigrated with the three subunits of heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB‘C and ABαC as well as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by the coelution of CAPP with trimeric and dimeric PP2A on size-exclusion chromatography. These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the biochemical investigation of CAPP. |
| doi_str_mv | 10.1021/bi980911+ |
| format | Article |
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In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purified to near homogeneity using hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS−polyacrylamide gel electrophoresis which comigrated with the three subunits of heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB‘C and ABαC as well as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by the coelution of CAPP with trimeric and dimeric PP2A on size-exclusion chromatography. 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These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the biochemical investigation of CAPP.</description><subject>Animals</subject><subject>Blotting, Western</subject><subject>Brain</subject><subject>Ceramides - physiology</subject><subject>Chromatography, Agarose</subject><subject>Chromatography, Gel</subject><subject>Chromatography, Ion Exchange</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme Activation - drug effects</subject><subject>Phosphoprotein Phosphatases - chemistry</subject><subject>Phosphoprotein Phosphatases - drug effects</subject><subject>Phosphoprotein Phosphatases - isolation & purification</subject><subject>Rats</subject><subject>Sepharose - analogs & derivatives</subject><subject>Sphingosine - analogs & derivatives</subject><subject>Sphingosine - pharmacology</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNplkN1LwzAUxYMoc04f_AOEPogIUk3SJk0eZ_ELpg6dH2_htk1Z5tbOJBX1r7fSuRefLvecH-dyD0L7BJ8STMlZZqTAkpCTDdQnjOIwlpJtoj7GmIdUcryNdpybtWuMk7iHepJLEXHZR6NxY01pcvCmrgKoiiCdgoXca2u-O7Eug1RbWJhCh8Pcmw_wugjGtvbaVMF4WrvlFDw47XbRVglzp_dWc4CeLi8m6XU4ur-6SYejEGJCfCjKDBhwHSdYZgnWJZY0T7KExbqIM8ZIIWgriUhmBQhSUMaAMco5L6Mo4SIaoKMud2nr90Y7rxbG5Xo-h0rXjVOifZsISlvwuANzWztndamW1izAfimC1W9z6q-5Fj1YZTbZQhdrcNVU64edb5zXn2sb7JviSZQwNRk_qpfzV57ePdyq55Y_7HjInZrVja3aRv6f_QER14Ky</recordid><startdate>19980811</startdate><enddate>19980811</enddate><creator>Galadari, Sehamuddin</creator><creator>Kishikawa, Katsuya</creator><creator>Kamibayashi, Craig</creator><creator>Mumby, Marc C</creator><creator>Hannun, Yusuf A</creator><general>American Chemical Society</general><scope>BSCLL</scope><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>7X8</scope></search><sort><creationdate>19980811</creationdate><title>Purification and Characterization of Ceramide-Activated Protein Phosphatases</title><author>Galadari, Sehamuddin ; Kishikawa, Katsuya ; Kamibayashi, Craig ; Mumby, Marc C ; Hannun, Yusuf A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a411t-8fba5a6e4709b70ef092c7b754ed4b551d82092839bda81d255a552666f337683</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Animals</topic><topic>Blotting, Western</topic><topic>Brain</topic><topic>Ceramides - physiology</topic><topic>Chromatography, Agarose</topic><topic>Chromatography, Gel</topic><topic>Chromatography, Ion Exchange</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme Activation - drug effects</topic><topic>Phosphoprotein Phosphatases - chemistry</topic><topic>Phosphoprotein Phosphatases - drug effects</topic><topic>Phosphoprotein Phosphatases - isolation & purification</topic><topic>Rats</topic><topic>Sepharose - analogs & derivatives</topic><topic>Sphingosine - analogs & derivatives</topic><topic>Sphingosine - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Galadari, Sehamuddin</creatorcontrib><creatorcontrib>Kishikawa, Katsuya</creatorcontrib><creatorcontrib>Kamibayashi, Craig</creatorcontrib><creatorcontrib>Mumby, Marc C</creatorcontrib><creatorcontrib>Hannun, Yusuf A</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Galadari, Sehamuddin</au><au>Kishikawa, Katsuya</au><au>Kamibayashi, Craig</au><au>Mumby, Marc C</au><au>Hannun, Yusuf A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and Characterization of Ceramide-Activated Protein Phosphatases</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1998-08-11</date><risdate>1998</risdate><volume>37</volume><issue>32</issue><spage>11232</spage><epage>11238</epage><pages>11232-11238</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Ceramide has emerged as a potential regulator of diverse cellular functions, and a few direct targets have been identified for its action including protein kinases and phosphatases. In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purified to near homogeneity using hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS−polyacrylamide gel electrophoresis which comigrated with the three subunits of heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB‘C and ABαC as well as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by the coelution of CAPP with trimeric and dimeric PP2A on size-exclusion chromatography. These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the biochemical investigation of CAPP.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>9698369</pmid><doi>10.1021/bi980911+</doi><tpages>7</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 1998-08, Vol.37 (32), p.11232-11238 |
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| language | eng |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | Animals Blotting, Western Brain Ceramides - physiology Chromatography, Agarose Chromatography, Gel Chromatography, Ion Exchange Electrophoresis, Polyacrylamide Gel Enzyme Activation - drug effects Phosphoprotein Phosphatases - chemistry Phosphoprotein Phosphatases - drug effects Phosphoprotein Phosphatases - isolation & purification Rats Sepharose - analogs & derivatives Sphingosine - analogs & derivatives Sphingosine - pharmacology |
| title | Purification and Characterization of Ceramide-Activated Protein Phosphatases |
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