Purification of the two forms of the high-molecular-weight neutral proteinase ingensin from rat liver.
Two forms of a high-molecular-weight proteinase were isolated from rat liver. The purification procedure involved homogenization of the tissue, chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC: TSK 3000 SWG) and hydroxyapatite chromatography. The breakthrough fraction from the hydroxyapatite column contained the sodium dodecyl sulphate (SDS)- and linoleic acid-activated proteinase, ingensin A, but the other form, ingensin B, which was also activated by SDS and linoleic acid, was bound to the hydroxyapatite and eluted at 200 mM phosphate. A distinct feature of ingensin A was its activation by a brief sonication procedure. The optimum pH of the two forms was 7.5-9.5, and both of them were activated by monovalent cations. Although both enzymes show similar molecular weights of 700,000 on gel filtration, ingensins A and B were separated into a major subunit of 120,000 and subunits of 25,000-35,000, respectively, under the denaturing conditions.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D017886:Durapatite; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification*; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011919:Rats, Inbred Strains; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D006882:Hydroxyapatites; D007527:Isoenzymes; D008041:Linoleic Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D012967:Sodium Dodecyl Sulfate; D017886:Durapatite; D019787:Linoleic Acid; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex