Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon
Distal colon absorbs K + through a Na +-independent, ouabain-sensitive H +/K +-exchange, associated to an apical ouabain-sensitive H +/K +-ATPase. Expression of HKα2, gene associated with this ATPase, induces K +-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensit...
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| Veröffentlicht in: | Archives of biochemistry and biophysics 2010-04, Vol.496 (1), p.21-32 |
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| creator | Belisario, Dimas C. Rocafull, Miguel A. del Castillo, Jesús R. |
| description | Distal colon absorbs K
+ through a Na
+-independent, ouabain-sensitive H
+/K
+-exchange, associated to an apical ouabain-sensitive H
+/K
+-ATPase. Expression of HKα2, gene associated with this ATPase, induces K
+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K
+-ATPase activities have been described in colonocytes. However, native H
+/K
+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H
+/K
+-ATPase from guinea-pig distal colon is described. H
+/K
+-ATPase is Mg
2+-dependent and activated by K
+, Cs
+ and NH
4
+ but not by Na
+ or Li
+, independently of K
+-accompanying anion. H
+/K
+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [
32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg
2+-dependent, vanadate-sensitive process. K
+ inhibited phosphorylation, effect blocked by ouabain. H
+/K
+-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na
+/K
+-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H
+/K
+-ATPase is a distinctive P-type ATPase. |
| doi_str_mv | 10.1016/j.abb.2010.01.014 |
| format | Article |
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+ through a Na
+-independent, ouabain-sensitive H
+/K
+-exchange, associated to an apical ouabain-sensitive H
+/K
+-ATPase. Expression of HKα2, gene associated with this ATPase, induces K
+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K
+-ATPase activities have been described in colonocytes. However, native H
+/K
+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H
+/K
+-ATPase from guinea-pig distal colon is described. H
+/K
+-ATPase is Mg
2+-dependent and activated by K
+, Cs
+ and NH
4
+ but not by Na
+ or Li
+, independently of K
+-accompanying anion. H
+/K
+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [
32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg
2+-dependent, vanadate-sensitive process. K
+ inhibited phosphorylation, effect blocked by ouabain. H
+/K
+-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na
+/K
+-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H
+/K
+-ATPase is a distinctive P-type ATPase.</description><identifier>ISSN: 0003-9861</identifier><identifier>EISSN: 1096-0384</identifier><identifier>DOI: 10.1016/j.abb.2010.01.014</identifier><identifier>PMID: 20122893</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Cell Membrane - metabolism ; Cell Polarity ; Colon - cytology ; Colon - enzymology ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic ; Guinea Pigs ; H(+)-K(+)-Exchanging ATPase - chemistry ; H(+)-K(+)-Exchanging ATPase - genetics ; H(+)-K(+)-Exchanging ATPase - isolation & purification ; H(+)-K(+)-Exchanging ATPase - metabolism ; HKα2 gene ; Isoenzymes - chemistry ; Isoenzymes - genetics ; Isoenzymes - isolation & purification ; Isoenzymes - metabolism ; Male ; Mammalian distal colon ; Molecular Sequence Data ; Mucous Membrane - cytology ; Na +/K +-ATPase β1-subunit ; Ouabain - metabolism ; Phosphoproteins - metabolism ; Phosphorylation ; Potassium absorption ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium - metabolism ; Tandem Mass Spectrometry</subject><ispartof>Archives of biochemistry and biophysics, 2010-04, Vol.496 (1), p.21-32</ispartof><rights>2010 Elsevier Inc.</rights><rights>2010 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c352t-67feb28375987a5b21fd87bc868f5aed525bbfe9ce60117c06b88776e44d80173</citedby><cites>FETCH-LOGICAL-c352t-67feb28375987a5b21fd87bc868f5aed525bbfe9ce60117c06b88776e44d80173</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.abb.2010.01.014$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20122893$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Belisario, Dimas C.</creatorcontrib><creatorcontrib>Rocafull, Miguel A.</creatorcontrib><creatorcontrib>del Castillo, Jesús R.</creatorcontrib><title>Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon</title><title>Archives of biochemistry and biophysics</title><addtitle>Arch Biochem Biophys</addtitle><description>Distal colon absorbs K
+ through a Na
+-independent, ouabain-sensitive H
+/K
+-exchange, associated to an apical ouabain-sensitive H
+/K
+-ATPase. Expression of HKα2, gene associated with this ATPase, induces K
+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K
+-ATPase activities have been described in colonocytes. However, native H
+/K
+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H
+/K
+-ATPase from guinea-pig distal colon is described. H
+/K
+-ATPase is Mg
2+-dependent and activated by K
+, Cs
+ and NH
4
+ but not by Na
+ or Li
+, independently of K
+-accompanying anion. H
+/K
+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [
32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg
2+-dependent, vanadate-sensitive process. K
+ inhibited phosphorylation, effect blocked by ouabain. H
+/K
+-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na
+/K
+-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H
+/K
+-ATPase is a distinctive P-type ATPase.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Cell Membrane - metabolism</subject><subject>Cell Polarity</subject><subject>Colon - cytology</subject><subject>Colon - enzymology</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>Guinea Pigs</subject><subject>H(+)-K(+)-Exchanging ATPase - chemistry</subject><subject>H(+)-K(+)-Exchanging ATPase - genetics</subject><subject>H(+)-K(+)-Exchanging ATPase - isolation & purification</subject><subject>H(+)-K(+)-Exchanging ATPase - metabolism</subject><subject>HKα2 gene</subject><subject>Isoenzymes - chemistry</subject><subject>Isoenzymes - genetics</subject><subject>Isoenzymes - isolation & purification</subject><subject>Isoenzymes - metabolism</subject><subject>Male</subject><subject>Mammalian distal colon</subject><subject>Molecular Sequence Data</subject><subject>Mucous Membrane - cytology</subject><subject>Na +/K +-ATPase β1-subunit</subject><subject>Ouabain - metabolism</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Potassium absorption</subject><subject>Rats</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Sodium - metabolism</subject><subject>Tandem Mass Spectrometry</subject><issn>0003-9861</issn><issn>1096-0384</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kEtLAzEUhYMoWqs_wI1k50KmTeaRZHAlxRcW7ELXIcncaMp0UpOZgv56I6MuhQuXeznnwPkQOqNkRgll8_VMaT3LSboJTVPuoQklNctIIcp9NCGEFFktGD1CxzGuCaG0ZPkhOkqWPBd1MUF2NQRnnVG98x1WXYPNmwrK9BDc5_j0FvdvgP2gtHJdFqGLrnc7wPf4cv6IL7Pr55WKgG3wG_w6uA5UtnWvuHGxVy02vvXdCTqwqo1w-rOn6OX25nlxny2f7h4W18vMFFXeZ4xb0LkoeFULriqdU9sIro1gwlYKmiqvtLZQG2CpCzeEaSE4Z1CWjSCUF1N0MeZug38fIPZy46KBtlUd-CFKXhS85pSLpKSj0gQfYwArt8FtVPiQlMhvunItE135TVcSmqZMnvOf9EFvoPlz_OJMgqtRAKnjzkGQ0TjoDDQugOll490_8V_pGYms</recordid><startdate>20100401</startdate><enddate>20100401</enddate><creator>Belisario, Dimas C.</creator><creator>Rocafull, Miguel A.</creator><creator>del Castillo, Jesús R.</creator><general>Elsevier Inc</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>7X8</scope></search><sort><creationdate>20100401</creationdate><title>Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon</title><author>Belisario, Dimas C. ; Rocafull, Miguel A. ; del Castillo, Jesús R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c352t-67feb28375987a5b21fd87bc868f5aed525bbfe9ce60117c06b88776e44d80173</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Cell Membrane - metabolism</topic><topic>Cell Polarity</topic><topic>Colon - cytology</topic><topic>Colon - enzymology</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Gene Expression Regulation, Enzymologic</topic><topic>Guinea Pigs</topic><topic>H(+)-K(+)-Exchanging ATPase - chemistry</topic><topic>H(+)-K(+)-Exchanging ATPase - genetics</topic><topic>H(+)-K(+)-Exchanging ATPase - isolation & purification</topic><topic>H(+)-K(+)-Exchanging ATPase - metabolism</topic><topic>HKα2 gene</topic><topic>Isoenzymes - chemistry</topic><topic>Isoenzymes - genetics</topic><topic>Isoenzymes - isolation & purification</topic><topic>Isoenzymes - metabolism</topic><topic>Male</topic><topic>Mammalian distal colon</topic><topic>Molecular Sequence Data</topic><topic>Mucous Membrane - cytology</topic><topic>Na +/K +-ATPase β1-subunit</topic><topic>Ouabain - metabolism</topic><topic>Phosphoproteins - metabolism</topic><topic>Phosphorylation</topic><topic>Potassium absorption</topic><topic>Rats</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Sodium - metabolism</topic><topic>Tandem Mass Spectrometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Belisario, Dimas C.</creatorcontrib><creatorcontrib>Rocafull, Miguel A.</creatorcontrib><creatorcontrib>del Castillo, Jesús R.</creatorcontrib><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>Archives of biochemistry and biophysics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Belisario, Dimas C.</au><au>Rocafull, Miguel A.</au><au>del Castillo, Jesús R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon</atitle><jtitle>Archives of biochemistry and biophysics</jtitle><addtitle>Arch Biochem Biophys</addtitle><date>2010-04-01</date><risdate>2010</risdate><volume>496</volume><issue>1</issue><spage>21</spage><epage>32</epage><pages>21-32</pages><issn>0003-9861</issn><eissn>1096-0384</eissn><abstract>Distal colon absorbs K
+ through a Na
+-independent, ouabain-sensitive H
+/K
+-exchange, associated to an apical ouabain-sensitive H
+/K
+-ATPase. Expression of HKα2, gene associated with this ATPase, induces K
+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K
+-ATPase activities have been described in colonocytes. However, native H
+/K
+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H
+/K
+-ATPase from guinea-pig distal colon is described. H
+/K
+-ATPase is Mg
2+-dependent and activated by K
+, Cs
+ and NH
4
+ but not by Na
+ or Li
+, independently of K
+-accompanying anion. H
+/K
+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [
32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg
2+-dependent, vanadate-sensitive process. K
+ inhibited phosphorylation, effect blocked by ouabain. H
+/K
+-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na
+/K
+-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H
+/K
+-ATPase is a distinctive P-type ATPase.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>20122893</pmid><doi>10.1016/j.abb.2010.01.014</doi><tpages>12</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-9861 |
| ispartof | Archives of biochemistry and biophysics, 2010-04, Vol.496 (1), p.21-32 |
| issn | 0003-9861 1096-0384 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_733797178 |
| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Amino Acid Sequence Animals Cell Membrane - metabolism Cell Polarity Colon - cytology Colon - enzymology Electrophoresis, Polyacrylamide Gel Gene Expression Regulation, Enzymologic Guinea Pigs H(+)-K(+)-Exchanging ATPase - chemistry H(+)-K(+)-Exchanging ATPase - genetics H(+)-K(+)-Exchanging ATPase - isolation & purification H(+)-K(+)-Exchanging ATPase - metabolism HKα2 gene Isoenzymes - chemistry Isoenzymes - genetics Isoenzymes - isolation & purification Isoenzymes - metabolism Male Mammalian distal colon Molecular Sequence Data Mucous Membrane - cytology Na +/K +-ATPase β1-subunit Ouabain - metabolism Phosphoproteins - metabolism Phosphorylation Potassium absorption Rats Reverse Transcriptase Polymerase Chain Reaction Sodium - metabolism Tandem Mass Spectrometry |
| title | Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon |
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