Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A
A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate: NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothr...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
1973-11-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520368437 |
| _version_ | 1818589892818501632 |
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| author | Frank H. Hulcher Wayne H. Oleson |
| author_facet | Frank H. Hulcher Wayne H. Oleson |
| author_sort | Frank H. Hulcher |
| collection | DOAJ |
| description | A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate: NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5′-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A–5,5′-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance–time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from εmax = 1.36 × 104 at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5–50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent Km value for 3-hydroxy-3-methylglutaryl CoA was 1.75 × 10–5 M, and for NADPH the value was 6.81 × 10–4 M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different. |
| first_indexed | 2024-12-16T09:47:52Z |
| format | Article |
| id | doaj.art-c8f876193c3944cab1a1acf6f076993e |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-16T09:47:52Z |
| publishDate | 1973-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-c8f876193c3944cab1a1acf6f076993e2022-12-21T22:36:07ZengElsevierJournal of Lipid Research0022-22751973-11-01146625631Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme AFrank H. Hulcher0Wayne H. Oleson1Department of Biochemistry and the Arteriosclerosis Research Center, The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103Department of Biochemistry and the Arteriosclerosis Research Center, The Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate: NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5′-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A–5,5′-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance–time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from εmax = 1.36 × 104 at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5–50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent Km value for 3-hydroxy-3-methylglutaryl CoA was 1.75 × 10–5 M, and for NADPH the value was 6.81 × 10–4 M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.http://www.sciencedirect.com/science/article/pii/S0022227520368437pigeon liveryeast microsomes3-hydroxy-3-methylglutaryl CoA deacylasesulfhydryl grouparsenite-dithiol complexenzyme kinetics |
| spellingShingle | Frank H. Hulcher Wayne H. Oleson Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A Journal of Lipid Research pigeon liver yeast microsomes 3-hydroxy-3-methylglutaryl CoA deacylase sulfhydryl group arsenite-dithiol complex enzyme kinetics |
| title | Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A |
| title_full | Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A |
| title_fullStr | Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A |
| title_full_unstemmed | Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A |
| title_short | Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A |
| title_sort | simplified spectrophotometric assay for microsomal 3 hydroxy 3 methylglutaryl coa reductase by measurement of coenzyme a |
| topic | pigeon liver yeast microsomes 3-hydroxy-3-methylglutaryl CoA deacylase sulfhydryl group arsenite-dithiol complex enzyme kinetics |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520368437 |
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