Diversity of rat brain cysteine proteinase inhibitors: isolation of low-molecular-weight cystatins and a higher-molecular weight T-kininogen-like glycoprotein.
Conditions for extraction of rat brain soluble and particulate cysteine proteinase inhibitors (CPIs) were compared and an optimal one was selected to isolate low- and high-molecular-weight forms active toward papain or brain cathepsins B/L. The different forms were purified by affinity chromatography on alkylated papain, and identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by use of Schiff's reagent, or by immunoblots using antisera to monomer or polymeric forms of human urinary cystatin c, to a human plasma histidine-rich glycoprotein (HRG), or to rat plasma T-kininogen. In particulates containing nuclei (P1) or synaptosomes (P2) the predominant CPI was an 80-kDa glycoprotein cross-reacting to anti-HRG and shown to be a T-kininogen by treatment with TPCK-trypsin, and subsequent bioassay of the released kinins. The levels found in rat brain were approximately 0.5 nmol/g wet weight. The higher-molecular-weight CPI potently inhibited cathepsin L hydrolysis of Leu-enkephalin at the Gly2-Gly3 bond with a Ki 10(-10) M. In contrast the low-molecular-weight CPIs were present in postmicrosomal fractions (S3) and cross-reacted with anti-cystatin c, but not with anti-HRG, anti-lysozyme, anti-beta protein amyloid peptide, or anti-T-kininogen. The low-molecular-weight forms were present at approximately 1-1.5 nmol/g wet weight and resembled "cerebrocystatin" purified previously from rat brain cytosol by M. Kopitar, F. Stern, and N. Marks [1983) Biochem. Biophys. Res. Commun. 112, 1000-1006.).
Marks
N
N
Center for Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, New York 10035.
Stern
F
F
Chi
L M
LM
Berg
M J
MJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D015853:Cysteine Proteinase Inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D007704:Kininogens / Q000302:isolation & purification*; D008297:Male; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000032:analysis
D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; D007704:Kininogens; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; C037791:cerebrocystatin