Evidence for the Presence of a Serine Proteinase(s) Associated with the Microsomal Membranes of Rat Liver1

Neutral proteinase activity in subcellular fractions of rat liver was measured with heat-denatured casein as a substrate before and after repeated freezing and thawing, followed by extraction of each fraction with 1 M KC1. By this treatment, most of the proteinase activity in each fraction was solubilized except for that in the microsomal fraction. More than 50 % of the neutral proteinase activity in the microsomal fraction remained insoluble, which indicated that the 1 M KCI-insoluble neutral proteinase activity was predominantly localized in the microsomal fraction. The activity was present in both the rough and smooth microsomes. The neutral proteinase(s) was largely solubilized from the 1 M KG-washed microsomal fraction by 0.5 M NaCl containing 0.5 % deoxycholate and 0.25 % cholate. The solubilized pro-teinase(s) was eluted from a Sepharose CL-6B column as a single but rather broad peak, giving a molecular weight of about 84,000. It showed maximal activity at pH 8.0 toward heat-de-natured casein as a substrate. It was markedly inhibited by diisopropyl phosphorofiuoridate, but was not significantly inhibited by chymostatin, soybean trypsin inhibitor, p-chloromer-curibenzoate or EDTA. o-Phenanthroline was somewhat inhibitory. Among urea-dena-tured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by casein and hemoglobin, whereas ovalbumin, bovine serum albumin, and 7"-globulin were not hydrolyzed to any significant extent. These results demonstrate that rat liver microsomes contain a unique serine proteinase(s) firmly bound to their membranes. In order to understand the mechanism of intracel- intracellular proteinases must be elucidated. Much lular protein breakdown and the roles of proteinases information has been accumulated to date on the therein, the distribution and properties of individual lysosomal proteinases and some other intracellular proteinases (2, 3). Most proteinases so far studie