Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography
A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [ 14C]HMG CoA was used as the substrate and the product formed, i.e., [ 14C]mevalonate, was allowed to be converted to its lac...
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| Veröffentlicht in: | Analytical biochemistry 1991-08, Vol.196 (2), p.211-214 |
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| creator | Ong, K.K. Khor, H.T. Tan, D.T.S. |
| description | A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [
14C]HMG CoA was used as the substrate and the product formed, i.e., [
14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05
m. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat. |
| doi_str_mv | 10.1016/0003-2697(91)90455-3 |
| format | Article |
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14C]HMG CoA was used as the substrate and the product formed, i.e., [
14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05
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14C]HMG CoA was used as the substrate and the product formed, i.e., [
14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05
m. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Anions</subject><subject>Biological and medical sciences</subject><subject>Chromatography, Ion Exchange - methods</subject><subject>Chromatography, Thin Layer - methods</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydroxymethylglutaryl CoA Reductases - isolation & purification</subject><subject>Hydroxymethylglutaryl CoA Reductases - metabolism</subject><subject>Mevalonic Acid - analogs & derivatives</subject><subject>Mevalonic Acid - isolation & purification</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kMFu1DAQhi0EKtvCG4CUA0Ll4GLHsR1fkFYroJUqcSlna9YZJ0ZJvNhJ1bw9WXZVbpzmMN__a-Yj5B1nN5xx9ZkxJmipjL42_JNhlZRUvCAbzoyiTDDzkmyekdfkMudfjHFeSXVBLrjWSimzIe02Z1iK6AtBu6VJ8Wmhgg44dUvf9vMEaemLXdwWCZvZTZCxADeFxzAtxZzD2BYwhjgGR_HJdTC2WLjYz8NYuC7FAabYJjh0yxvyykOf8e15XpGf374-7G7p_Y_vd7vtPXWiVhOtygq8q0EpxAahMhr2qnZSN6BEyQ1ztaxYs2cSdMmUKve-8bquJJRec-_FFfl46j2k-HvGPNkhZId9DyPGOVtdKiEM1ytYnUCXYs4JvT2kMKzvWs7s0a89yrNHedZw-9evFWvs_bl_3g_Y_AudhK77D-c9ZAe9TzC6kJ8xWZaVVnLFvpwwXF08Bkw2u4CjwyYkdJNtYvj_HX8A4paYMw</recordid><startdate>19910801</startdate><enddate>19910801</enddate><creator>Ong, K.K.</creator><creator>Khor, H.T.</creator><creator>Tan, D.T.S.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</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>19910801</creationdate><title>Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography</title><author>Ong, K.K. ; Khor, H.T. ; Tan, D.T.S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-424afc8a66eedea497ab68c57da632190c8540db05a720662bfdf7845a2f71ff3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Anions</topic><topic>Biological and medical sciences</topic><topic>Chromatography, Ion Exchange - methods</topic><topic>Chromatography, Thin Layer - methods</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hydroxymethylglutaryl CoA Reductases - isolation & purification</topic><topic>Hydroxymethylglutaryl CoA Reductases - metabolism</topic><topic>Mevalonic Acid - analogs & derivatives</topic><topic>Mevalonic Acid - isolation & purification</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ong, K.K.</creatorcontrib><creatorcontrib>Khor, H.T.</creatorcontrib><creatorcontrib>Tan, D.T.S.</creatorcontrib><collection>Pascal-Francis</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>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ong, K.K.</au><au>Khor, H.T.</au><au>Tan, D.T.S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1991-08-01</date><risdate>1991</risdate><volume>196</volume><issue>2</issue><spage>211</spage><epage>214</epage><pages>211-214</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A rapid, easy, and sensitive method is described in this paper for the assay of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, a key enzyme in cholesterol biosynthesis. [
14C]HMG CoA was used as the substrate and the product formed, i.e., [
14C]mevalonate, was allowed to be converted to its lactone form (mevalonolactone) in the presence of HCl. The reaction mixture was applied to a column containing an anionic exchanger. The column was made up of QAE-Sephadex (A25, formate form) packed to a height of 4 cm in Pasteur pipets. Under these conditions, mevalonolactone was not retained by the column and was eluted with ammonium formate solution while HMG CoA, being negatively charged, was retained by the gel and eluted by HCl above 0.05
m. Determination of the amount of radioactivity in mevalonolactone was then used to quantitate the activity of HMG CoA reductase. This assay has been successfully used for determining the activity of this enzyme in a microsomal fraction prepared from the liver of the rat.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>1776669</pmid><doi>10.1016/0003-2697(91)90455-3</doi><tpages>4</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 1991-08, Vol.196 (2), p.211-214 |
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| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Analytical, structural and metabolic biochemistry Anions Biological and medical sciences Chromatography, Ion Exchange - methods Chromatography, Thin Layer - methods Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Hydroxymethylglutaryl CoA Reductases - isolation & purification Hydroxymethylglutaryl CoA Reductases - metabolism Mevalonic Acid - analogs & derivatives Mevalonic Acid - isolation & purification |
| title | Assay of 3-hydroxy-3-methylglutaryl CoA reductase activity using anionic-exchange column chromatography |
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