PURIFICATION AND CHARACTERIZATION OF HUMAN BRAIN RIBONUCLEASE INHIBITOR

Ribonuclease inhibitor (RI) was purified about 1300-fold from human ce rebrum (including a small portion of midbrain) by a combination of amm onium sulfate precipitation, ribonuclease A-Sepharose chromatography, and high-performance anion-exchange chromatography. The purified RI ap peared to be homogeneous as judged by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). Using the same method, a homogen eous RI was also obtained from human hindbrain (brainstem and cerebell um). The cerebral RI appeared to be virtually identical with the hindb rain RI on the basis of the following properties: (a) Molecular mass w as estimated to be 50 kDa on SDS-PAGE under reducing conditions. (b) C omposition analysis revealed that the RI was rich in leucine and cyste ine residues and included no amino sugars. (c) The N-terminus was bloc ked and probably modified by N-acetylation. After treatment with trifl uoroacetic acid, it became susceptible to Edman degradation and was se quenced as eu-Asp-Ile-Gln-Ser-Leu-Asp-Ile-Gln-(Cys)-Glu-Glu-. (d) The RI, which showed sulfhydryl-dependent inhibitory activity on both secr etory-type and nonsecretory-type ribonucleases, bound tightly to ribon uclease to form a 1:1 complex on a molar basis. (e) The RI cross-react ed strongly with anti-human placental RI antibody. These findings also indicate that human brain RI is quite similar to human placental RI. In contrast to the abundance of RI in human brain tissue (about 0.08% (w/w) of total protein), RI was undetectable in human cerebrospinal fl uid, suggesting that brain RI may not be a secreted protein. (C) 1994 Academic Press, Inc.