Purification and partial characterization of a cytosolic proteinase from rat liver

Abstract

A cytosolic proteinase has been purified to near homogeniety from rat liver. The clear supernatant obtained from a rat liver homogenate was treated with DEAE-cellulose and proteinase activity eluted by batch elution between 0.12-0.5 M KCl. Further purification was achieved using molecular sieve chromatography (Ultragel AcA22) and ion exchange chromatography (DEAE-cellulose, 0-0.3 M KCl). The proteinase has a molecular weight of 576,000 and an isoelectric point of 4.9-5.0. The proteinase is a very specific enzyme. It does not hydrolyze aminopeptidase substrates or TAME. Low levels of activity are observed using BTEE as substrate. The proteinase does not hydrolyze the oxidized B-chain of insulin, lysozyme, or cytochrome c. Globin and (beta)-casein are hydrolyzed by the proteinase and the hydrolysis of (beta)-casein results in the formation of three large fragments. Proteinase activity is inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor and TPCK but not by TLCK. Leupeptin, pepstatin and elastatinal did not inhibit proteinase activity. Chymostatin and antipain inhibit the proteinase. The results obtained using inhibitors suggest that the proteinase is chymotrypsin-like. Soluble cell proteins obtained from Reuber H-35 cells were labeled to high specific activity and fractionated by molecular sieve chromatography, isoelectric focusing and hydrophobic chromatography. These parameters were chosen because the characteristics of molecular weight, isoelectric point and surface hydrophobicity are known to correlate with turnover rates in vivo. No relationship was observed between proteolytic susceptibility to the proteinase and characteristics associated with short half-lives in vivo. A relationship between proteolytic susceptibility and short half-lives in vivo was demonstrated with trypsin and subtilisin. Proteins which are more hydrophobic, larger proteins, and acidic proteins are rapidly turning over in vivo and are more susceptible to proteolysis by trypsin and subtilisin. The function of the proteinase is not known; however, the limited ability to hydrolyze proteins suggests a specific role for this enzyme. Specific, limited cleavage of proteins could be an initial event in the degradation of intracellular proteins by a non-lysosomal pathway. Alternatively, the specificity of the proteinase is consistent with a role in protein processing.