Ribonuclease inhibitor from pig brain: purification, characterization, and direct spectrophotometric assay.
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.
Cho
S W
SW
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Joshi
J G
JG
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D005260:Female; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D010926:Placental Hormones; D011247:Pregnancy; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D013053:Spectrophotometry / Q000379:methods; D013552:Swine
D000596:Amino Acids; D006882:Hydroxyapatites; D009419:Nerve Tissue Proteins; D010926:Placental Hormones; C022052:placental ribonuclease inhibitor; D017886:Durapatite; D012260:Ribonucleases