Ribonuclease inhibitor from pig brain: purification, characterization, and direct spectrophotometric assay |
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Authors: | S W Cho J G Joshi |
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Institution: | Department of Biochemistry, University of Tennessee, Knoxville 37996-0840. |
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Abstract: | The ribonuclease inhibitor from pig brain has been purified 1,500-fold by a combination of ammonium sulfate fractionation, ion-exchange chromatography, hydroxylapatite chromatography, and gel filtration. The inhibitor has a Mr 50,000. It is a noncompetitive inhibitor for pancreatic ribonuclease A with a Ki of 1 nM, forming a 1:1 complex. Both ribonuclease A and B, but not ribonuclease U1 and T1, are inactivated by the inhibitor. The inhibition capacity was abolished by sulfhydryl reagents such as p-chloromercuribenzoate. Incubation of the enzyme-inhibitor complex with the sulfhydryl reagent caused dissociation into active ribonuclease and inactive inhibitor. Dithiothreitol was required during purification to retain the activity of the inhibitor. |
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