Purification and properties of a protein from bovine lens which inhibits trypsin and two endogenous lens proteinases.
An inhibitor of trypsin-like proteinases was isolated from the water-soluble proteins of bovine lens cortex. The inhibitor was purified by four simple procedures: the separation of the inhibitor fraction by Agarose A-1.5 m gel filtration, extraction with 2.5% TCA at 70 degrees C, ammonium sulfate precipitation of the TCA-soluble proteins and a final separation by gel filtration chromatography. This preparation was found to be homogeneous by SDS-PAGE with an approximate subunit molecular weight of 5500 daltons. Gel filtration separated the ammonium sulfate precipitate into an inactive high-molecular-weight peak which eluted in the void volume, and two peaks of approximately 40 000 daltons and 10 000 daltons. Both low-molecular-weight peaks gave a single 5500 dalton band on SDS-PAGE, but only the 40 000 dalton peak was active when concentrated and assayed with bovine trypsin. These data suggest that the inhibitor is present in multimeric forms in solution, but only the octamer appears to be active. Antibodies prepared against the purified inhibitor showed a single precipitin line, while no reaction was seen with an alpha-crystallin antiserum. Upon storage in solution all of the inhibitor became converted into a high-molecular-weight form which was completely inactive. SDS-PAGE dissociated the inhibitor aggregate into a major 44 000 dalton band along with several minor bands. Amino acid analysis showed that the purified inhibitor contains a very high content of hydrophobic residues. The lens inhibitor was effective in reducing the activity of trypsin, but complete inhibition was not seen even at high inhibitor levels. A rapid and complete inhibition was observed, however, with two endogenous trypsin-like proteinases isolated from the alpha-crystallin region.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000302:isolation & purification*; D005260:Female; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007904:Lens Cortex, Crystalline / Q000032:analysis* / Q000201:enzymology; D007908:Lens, Crystalline / Q000032:analysis*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000302:isolation & purification*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000302:isolation & purification*
D000596:Amino Acids; D003459:Crystallins; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D014361:Trypsin Inhibitors