D. Nadano et al., PURIFICATION AND CHARACTERIZATION OF HUMAN BRAIN RIBONUCLEASE INHIBITOR, Archives of biochemistry and biophysics, 312(2), 1994, pp. 421-428
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.