Purification and characterization of human brain ribonuclease inhibitor.
Ribonuclease inhibitor (RI) was purified about 1300-fold from human cerebrum (including a small portion of midbrain) by a combination of ammonium sulfate precipitation, ribonuclease A-Sepharose chromatography, and high-performance anion-exchange chromatography. The purified RI appeared to be homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using the same method, a homogeneous RI was also obtained from human hindbrain (brainstem and cerebellum). The cerebral RI appeared to be virtually identical with the hindbrain RI on the basis of the following properties: (a) Molecular mass was estimated to be 50 kDa on SDS-PAGE under reducing conditions. (b) Composition analysis revealed that the RI was rich in leucine and cysteine residues and included no amino sugars. (c) The N-terminus was blocked and probably modified by N-acetylation. After treatment with trifluoroacetic acid, it became susceptible to Edman degradation and was sequenced as Ser-Leu-Asp-Ile-Gln-Ser-Leu-Asp-Ile-Gln-(Cys)-Glu-Glu-. (d) The RI, which showed sulfhydryl-dependent inhibitory activity on both secretory-type and nonsecretory-type ribonucleases, bound tightly to ribonuclease to form a 1:1 complex on a molar basis. (e) The RI cross-reacted 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 fluid, suggesting that brain RI may not be a secreted protein.
Nadano
D
D
Department of Legal Medicine, Fukui Medical School, Japan.
Yasuda
T
T
Takeshita
H
H
Uchide
K
K
Kishi
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001923:Brain Chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D017421:Sequence Analysis; D014018:Tissue Distribution
D000596:Amino Acids; D009419:Nerve Tissue Proteins; C088463:ribonuclease inhibitor, brain; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic