Purification of human kidney angiotensin I converting enzyme using reverse-immunoadsorption chromatography |
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Authors: | John A Weare John T Gafford HS Lu Ervin G Erdös |
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Institution: | 1. Department of Pharmacology, University of Texas Health Science Center, Dallas, Texas 75235 USA;2. Department of Internal Medicine, University of Texas Health Science Center, Dallas, Texas 75235 USA;3. Department of Chemistry, North Texas State University, Denton, Texas 76203 USA;4. Department of Biochemistry, North Texas State University, Denton, Texas 76203 USA |
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Abstract: | A rapid and highly efficient procedure for purification of angiotensin I converting enzyme from human kidney has been developed. Following tryptic solubilization, the enzyme was partially purified by DEAE-cellulose and hydroxylapatite chromatography. The final step consisted of “reverse immunoadsorption” on a column prepared by coupling antisera raised against contaminating proteins to CNBr-activated Sepharose CL-6B. Starting with 600 g kidney tissue, 6.1 mg of enzyme was obtained with a specific activity of 108 U/mg using Hip-His-Leu as substrate, a 3400-fold purification with an overall yield of 26%. The preparation gave a single band on 7.5% SDS-urea gels and a single arc against antisera to impure enzyme in crossed immunoelectrophoresis. A single N-terminal amino acid (leucine) was detected by dansylation. This procedure has allowed the initiation of structural studies with the human enzyme. “Reverse immunoadsorption” may be a generally useful method for protein purification. |
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Keywords: | CE angiotensin I converting enzyme Hip hippuryl benzoyl-glycyl Hepes SDS sodium dodecyl sulfate |
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